Injectable neurosteroid formulations containing nanoparticles

ABSTRACT

The disclosure provides an injectable neurosteroid nanoparticle formulation comprising nanoparticles having a D50 of less than 2000 nm the nanoparticles comprising a neurosteroid of Formula I, 
     
       
         
         
             
             
         
       
     
     where the variables R 1 -R 9  and X are defined herein and at least one surface stabilizer. The surface stabilizer can be a polymeric surface stabilizer such as hydroxyethyl starch, dextran, or povidone. The injectable neurosteroid nanoparticle formulation can be an intravenous formulation. The disclosure also provides a lyophilized powder of the injectable neurosteroid nanoparticle formulation that can be reconstituted in an aqueous solution prior to administration. The disclosure provides injectable neurosteroid nanoparticle formulations and dry powders of such formulations that have been sterilized by ebeam irradiation. The disclosure provides a method of treating a patient having a seizure disorder, stroke, or traumatic brain injury, comprising administering an effective amount of the injectable neurosteroid nanoparticle formulation. The disclosure also provides combination methods in which the injectable neurosteroid nanoparticle formulation is a first active agent that is administered in combination with at least one additional active agent.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/294,135, filed Oct. 14, 2016, which claims the benefit of U.S.Provisional Application No. 62/242,601, filed on Oct. 16, 2015, each ofwhich are incorporated by reference in their entirety.

BACKGROUND

Pregnane neurosteroids are a class of compounds useful as anesthetics,sedatives, hypnotics, anxiolytics, and anticonvulsants. These compoundsare marked by very low aqueous solubility, which limits theirformulation options. Injectable formulations of pregnane neurosteroidsare particularly desirable as these compounds are used for clinicalindications for which oral administration is precluded, such asanesthesia and particularly for the treatment of active seizures.

Status epilepticus (SE) is a serious seizure disorder in which theepileptic patient experiences a seizure lasting more than five minutes,or more than one seizure in a five minute period without recoveringbetween seizures. In certain instances convulsive seizures may last daysor even weeks. Status epilepticus is treated in the emergency room withconventional anticonvulsants. GABA_(A) receptor modulators such asbenzodiazepines (BZs) are a first line treatment. Patients who fail torespond to BZs alone are usually treated with anesthetics orbarbiturates in combination with BZs. About 23-43% of status epilepticuspatients who are treated with a benzodiazepine and at least oneadditional antiepileptic drug fail to respond to treatment and areconsidered refractory (Rossetti, A. O. and Lowenstein, D. H., LancetNeurol. (2011) 10(10): 922-930.) There are currently no good treatmentoptions for these patients. The mortality rate for refractory statusepilepticus (RSE) patients is high and most RSE patients do not returnto their pre-RSE clinical condition. About 15% of patients admitted tohospital with SE are in a subgroup of RSE patients said to besuper-refractory SE (SRSE), in which the patients have continued orrecurrent seizures 24 hours or more after the onset of anesthetictherapy. SRSE is associated with high rates of mortality and morbidity.(Shorvon, S., and Ferlisi, M., Brain, (2011) 134(10): 2802-2818.)

Another serious seizure disorder is PCDH19 female pediatric epilepsy,which affects approximately 15,000-30,000 females in the United States.This genetic disorder is associated with seizures beginning in the earlyyears of life, mostly focal clustered seizures that can last for weeks.The mutation of the PCDH19 gene has been associated with low levels ofallopregnanolone. Currently there are no approved therapies for PCDH19female pediatric epilepsy.

Thus, there exists the need for additional treatments for seizuredisorders such as status epilepticus, refractory status epilepticus,super refractory status epilepticus, and PCDH19 female pediatricepilepsy. This disclosure fulfills this need by providing injectablepregnane neurosteroid formulations and provides additional advantagesthat are described herein.

SUMMARY

The disclosure provides an injectable nanoparticle pregnane neurosteroidformulation comprising nanoparticles having a D50 (volume weightedmedian diameter) of less than 2000 nm (nanometers) and the nanoparticlescomprising a pregnane neurosteroid, at least one surface stabilizer, forexample a polymer surface stabilizer such as hydroxyethyl starch,dextran, or povidone, and in some embodiment an additional surfacestabilizer, such as a surfactant. An embodiment of the formulationcomprises the nanoparticles in an aqueous suspension. The disclosurealso provides a lyophilized powder of the pregnane neurosteroidnanoparticle formulation that may be reconstituted in water forinjection.

The disclosure provides a neurosteroid formulation comprisingnanoparticles having a D50 of less than 2000 nm, the nanoparticlescomprising

a) a neurosteroid of Formula I:

or a pharmaceutically acceptable salt thereof, wherein:

X is O, S, or NR¹⁰;

R¹ is hydrogen, hydroxyl, optionally substituted alkyl, optionallysubstituted heteroalkyl, optionally substituted aryl, or optionallysubstituted arylalkyl;

R⁴ is hydrogen, hydroxyl, oxo, optionally substituted alkyl, oroptionally substituted heteroalkyl,

R², R³, R⁵, R⁶, and R⁷ are each independently hydrogen, hydroxyl,halogen, optionally substituted alkyl, or optionally substitutedheteroalkyl;

R⁸ is hydrogen or alkyl and R⁹ is hydroxyl; or

R⁸ and R⁹ are taken together to form an oxo group;

R¹⁰ is hydrogen, hydroxyl, optionally substituted alkyl, optionallysubstituted heteroalkyl, optionally substituted aryl, or optionallysubstituted arylalkyl where

each alkyl is a C₁-C₁₀alkyl, C₃-C₆cycloalkyl,(C₃-C₆cycloalkyl)C₁-C₄alkyl, and optionally contains a single bondreplaced by a double or triple bond;

each heteroalkyl group is an alkyl group in which one or more methylgroup is replaced by an independently chosen —O—, —S—, —N(R¹⁰)—, —S(═O)—or —S(═O)₂—, where R¹⁰ is hydrogen, alkyl, or alkyl in which one or moremethylene group is replaced by —O—, —S—, —NH, or —N-alkyl; and

b) at least one surface stabilizer.

The disclosure also includes embodiments of the above injectableneurosteroid nanoparticle formulation in which the nanoparticles have aD50 of less than 500 which contain a surfactant as an additional surfacestabilizer. The disclosure also includes neurosteroid nanoparticleshaving a D50 of less than 500 nm, the nanoparticles comprising

a) a compound or salt of Formula I;

b) a polymeric surface stabilizer; and

c) at least one additional surface stabilizer, wherein the additionalsurface stabilizer is a surfactant.

In certain embodiments the neurosteroid is ganaxolone (GNX) orallopregnanolone (ALLO). In certain embodiments the neurosteroid isganaxolone.

The disclosure also provides a method of treating a patient having aseizure disorder, stroke, or traumatic brain injury, comprisingadministering an effective amount of the injectable neurosteroidnanoparticle formulation comprising a neurosteroid of Formula I (e.g.ganaxolone or allopregnanolone), either hydroxyethyl starch, dextran, orpovidone, and a surfactant, in the form of nanoparticles; and water.

The disclosure includes methods of treatment in which the neurosteroidis the only active agent and methods in which the neurosteroid, of theneurosteroid nanoparticle formulation, is a first active agent and isadministered in combination with an additional active agent.

The disclosure includes methods of treatment which includeadministration schedules for the neurosteroid nanoparticle formulation,in which the neurosteroid is the only active agent or in which themethod includes treatment with at least one additional active agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . D50 values of a ganaxolone nanosuspension stabilized byhydroxyethyl starch and sodium deoxycholate monitored over a 17-dayperiod. The nanosuspension contained ganaxolone (20%), hydroxyethylstarch (6%), sodium deoxycholate (1.2%), and simethicone (0.06%). Theparticle size was substantially unchanged over the period monitored.

FIG. 2 . D50 values of a ganaxolone nanosuspension stabilized bypoloxamer 188 and sodium deoxycholate monitored over a 6-day period. Thenanosuspension contained ganaxolone (10%), poloxamer 188 (12.5%),dextran (40K MW) (5%), sodium deoxycholate (0.25%).

FIGS. 3A-3C. Mean plasma ganaxolone concentration (ng/mL) for 2 hoursfor ganaxolone hydroxyethyl starch formulation and positive controlganaxolone Captisol formulation in rats after a single intravenousinjection, for (A) 9 mg/kg, (B) 12 mg/kg, and (C) 15 mg/kg doses.

FIGS. 4A-4C. Mean brain ganaxolone concentration (ng/mL). for 2 hoursfor ganaxolone hydroxyethyl starch formulation and positive controlganaxolone Captisol formulation in rats after a single intravenousinjection, for (A) 9 mg/kg, (B) 12 mg/kg, and (C) 15 mg/kg doses.

FIGS. 5A-5B. (A) Ganaxolone brain and plasma levels in rats receivingintravenous Ganaxolone as a Captisol solution or a nanosuspension. (B)Allopregnanolone brain levels, in rats. Experiment performed as forGanaxolone.

FIGS. 6A-6D. Particle size distribution curves for particles containing(A) ganaxolone and hydroxyethyl starch (D50=106 nm), (B) ganaxolone andDextran 70 (D50=111 nm), (C) ganaxolone and povidone (D50=109 nm), and(D) allopregnanolone and hydroxyethyl starch (D50=96 nm).

FIGS. 7A-7C. Behavior scores. for 4 hours for ganaxolone nanoparticlehydroxyethyl starch formulation and positive control ganaxolone Captisolformulation in rats after a single intravenous injection, for (A) 9mg/kg, (B) 12 mg/kg, and (C) 15 mg/kg doses.

DETAILED DESCRIPTION Definitions

Recitation of ranges of values are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. The endpoints of all ranges are includedwithin the range and independently combinable. All methods describedherein can be performed in a suitable order unless otherwise indicatedherein or otherwise clearly contradicted by context. The use of any andall examples, or exemplary language (e.g., “such as”), is intendedmerely for illustration and does not pose a limitation on the scope ofthe invention unless otherwise claimed. No language in the specificationshould be construed as indicating any non-claimed element as essentialto the practice of the invention.

The terms “a” and “an” do not denote a limitation of quantity, butrather denote the presence of at least one of the referenced item.

The term “about” is used synonymously with the term “approximately.” Asone of ordinary skill in the art would understand, the exact boundary of“about” will depend on the component of the composition. Illustratively,the use of the term “about” indicates that values slightly outside thecited values, i.e., plus or minus 0.1% to 10%, which are also effectiveand safe. Thus compositions slightly outside the cited ranges are alsoencompassed by the scope of the present claims.

An “active agent” is any compound, element, or mixture that whenadministered to a patient alone or in combination with another agentconfers, directly or indirectly, a physiological effect on the patient.When the active agent is a compound, salts, solvates (includinghydrates) of the free compound or salt, crystalline and non-crystallineforms, as well as various polymorphs of the compound are included.Compounds may contain one or more asymmetric elements such asstereogenic centers, stereogenic axes and the like, e.g. asymmetriccarbon atoms, so that the compounds can exist in differentstereoisomeric forms. These compounds can be, for example, racemates oroptically active forms. For compounds with two or more asymmetricelements, these compounds can additionally be mixtures of diastereomers.For compounds having asymmetric centers, it should be understood thatall of the optical isomers in pure form and mixtures thereof areencompassed. In addition, compounds with carbon-carbon double bonds mayoccur in Z- and E-forms, with all isomeric forms of the compounds beingincluded in the present invention. In these situations, the singleenantiomers, i.e. optically active forms, can be obtained by asymmetricsynthesis, synthesis from optically pure precursors, or by resolution ofthe racemates. Resolution of the racemates can also be accomplished, forexample, by conventional methods such as crystallization in the presenceof a resolving agent, or chromatography, using, for example a chiralHPLC column.

The terms “comprising,” “including,” and “containing” are non-limiting.Other non-recited elements may be present in embodiments claimed bythese transitional phrases. Where “comprising,” “containing,” or“including” are used as transitional phrases other elements may beincluded and still form an embodiment within the scope of the claim. Theopen-ended transitional phrase “comprising” encompasses the intermediatetransitional phrase “consisting essentially of” and the close-endedphrase “consisting of.”

“Alkyl” is a branched or straight chain saturated aliphatic hydrocarbongroup, having the specified number of carbon atoms, generally from 1 toabout 8 carbon atoms. The term C₁-C₆-alkyl as used herein indicates analkyl group having from 1, 2, 3, 4, 5, or 6 carbon atoms. Otherembodiments include alkyl groups having from 1 to 6 carbon atoms, 1 to 4carbon atoms or 1 or 2 carbon atoms, e.g. C₁-C₈-alkyl, C₁-C₄-alkyl, andC₁-C₂-alkyl. Examples of alkyl include, but are not limited to, methyl,ethyl, n-propyl, isopropyl, n-butyl, 3-methylbutyl, t-butyl, n-pentyl,and sec-pentyl.

“Aryl” indicates aromatic groups containing only carbon in the aromaticring or rings. Typical aryl groups contain 1 to 3 separate, fused, orpendant rings and from 6 to about 18 ring atoms, without heteroatoms asring members. When indicated, such aryl groups may be furthersubstituted with carbon or non-carbon atoms or groups. Aryl groupsinclude, for example, phenyl, naphthyl, including 1-naphthyl,2-naphthyl, and bi-phenyl. An “arylalkyl” substituent group is an arylgroup as defined herein, attached to the group it substitutes via analkylene linker. The alkylene is an alkyl group as described hereinexcept that it is bivalent.

A “bolus dose” is a relatively large dose of medication administered ina short period, for example within 1 to 30 minutes.

C_(max) is the measured concentration of an active concentration in theplasma at the point of maximum concentration.

“Cycloalkyl” is a saturated hydrocarbon ring group, having the specifiednumber of carbon atoms. Monocyclic cycloalkyl groups typically have from3 to about 8 carbon ring atoms or from 3 to 6 (3, 4, 5, or 6) carbonring atoms. Cycloalkyl substituents may be pendant from a substitutednitrogen, oxygen, or carbon atom, or a substituted carbon atom that mayhave two substituents may have a cycloalkyl group, which is attached asa spiro group. Examples of cycloalkyl groups include cyclopropyl,cyclobutyl, cyclopentyl, and cyclohexyl.

A “heteroalkyl” group is an alkyl group as described with at least onecarbon replaced by a heteroatom, e.g. N, O, or S.

“Infusion” administration is a non-oral administration, typicallyintravenous though other non-oral routes such as epidural administrationare included in some embodiments. Infusion administration occurs over alonger period than a bolus administration, for example over a period ofat least 15 minutes, at least 30 minutes, at least 1 hour, at least 2hours, at least 3 hours, or at least 4 hours.

A “patient” is a human or non-human animal in need of medical treatment.Medical treatment includes treatment of an existing condition, such as adisorder or injury. In certain embodiments treatment also includesprophylactic or preventative treatment, or diagnostic treatment.

“Pharmaceutical compositions” are compositions comprising at least oneactive agent, such as a compound or salt, solvate, or hydrate of Formula(I), and at least one other substance, such as a carrier. Pharmaceuticalcompositions optionally contain one or more additional active agents.When specified, pharmaceutical compositions meet the U.S. FDA's GMP(good manufacturing practice) standards for human or non-human drugs.“Pharmaceutical combinations” are combinations of at least two activeagents which may be combined in a single dosage form or providedtogether in separate dosage forms with instructions that the activeagents are to be used together to treat a disorder, such as a seizuredisorder.

“Povidone” also known as polyvidone and polyvinylpyrrolidone (PVP) is awater soluble polymer made from the monomer, N-vinylpyrrolidone.Plasdone C-12 and C-17 are pharmaceutical grade homopolymers ofN-vinylpyrrolidone. Plasdone C-12 has a K value of 10-2-13.8 and nominalmolecular weight of 4000 d. Plasdone C-17 has a K-value of 15.5-17.5 andnominal molecular weight of 10,000 d.

The term “substituted” as used herein, means that any one or morehydrogens on the designated atom or group is replaced with a selectionfrom the indicated group, provided that the designated atom's normalvalence is not exceeded. When the substituent is oxo (i.e., ═O) then 2hydrogens on the atom are replaced. When an oxo group substitutes aheteroaromatic moiety, the resulting molecule can sometimes adopttautomeric forms. For example a pyridyl group substituted by oxo at the2- or 4-position can sometimes be written as a pyridine orhydroxypyridine. Combinations of substituents and/or variables arepermissible only if such combinations result in stable compounds oruseful synthetic intermediates. A stable compound or stable structure ismeant to imply a compound that is sufficiently robust to surviveisolation from a reaction mixture and subsequent formulation into aneffective therapeutic agent. Unless otherwise specified, substituentsare named into the core structure. For example, it is to be understoodthat aminoalkyl means the point of attachment of this substituent to thecore structure is in the alkyl portion and alkylamino means the point ofattachment is a bond to the nitrogen of the amino group.

Suitable groups that may be present on a “substituted” or “optionallysubstituted” position include, but are not limited to, e.g., halogen;cyano; —OH; oxo; —NH₂; nitro; azido; alkanoyl (such as a C₂-C₆ alkanoylgroup); C(O)NH₂; alkyl groups (including cycloalkyl and(cycloalkyl)alkyl groups) having 1 to about 8 carbon atoms, or 1 toabout 6 carbon atoms; alkenyl and alkynyl groups including groups havingone or more unsaturated linkages and from 2 to about 8, or 2 to about 6carbon atoms; alkoxy groups having one or more oxygen linkages and from1 to about 8, or from 1 to about 6 carbon atoms; aryloxy such asphenoxy; alkylthio groups including those having one or more thioetherlinkages and from 1 to about 8 carbon atoms, or from 1 to about 6 carbonatoms; alkylsulfinyl groups including those having one or more sulfinyllinkages and from 1 to about 8 carbon atoms, or from 1 to about 6 carbonatoms; alkylsulfonyl groups including those having one or more sulfonyllinkages and from 1 to about 8 carbon atoms, or from 1 to about 6 carbonatoms; aminoalkyl groups including groups having one or more N atoms andfrom 1 to about 8, or from 1 to about 6 carbon atoms; mono- ordialkylamino groups including groups having alkyl groups from 1 to about6 carbon atoms; mono- or dialkylaminocarbonyl groups (i.e. alkylNHCO— or(alkyl1)(alkyl2)NCO—) having alkyl groups from about 1 to about 6 carbonatoms; aryl having 6 or more carbons.

“Sterilize” means to inactivate substantially all biologicalcontaminates in a sample, formulation, or product. A 1-million foldreduction in the bioburden is also considered “sterilized” for mostpharmaceutical applications.

A “therapeutically effective amount” or “effective amount” is thatamount of a pharmaceutical agent to achieve a pharmacological effect.The term “therapeutically effective amount” includes, for example, aprophylactically effective amount. An “effective amount” of neurosteroidis an amount needed to achieve a desired pharmacologic effect ortherapeutic improvement without undue adverse side effects. Theeffective amount of neurosteroid will be selected by those skilled inthe art depending on the particular patient and the disease. It isunderstood that “an effective amount” or “a therapeutically effectiveamount” can vary from subject to subject, due to variation in metabolismof neurosteroid, age, weight, general condition of the subject, thecondition being treated, the severity of the condition being treated,and the judgment of the prescribing physician.

“Treat” or “treatment” refers to any treatment of a disorder or disease,such as inhibiting the disorder or disease, e.g., arresting thedevelopment of the disorder or disease, relieving the disorder ordisease, causing regression of the disorder or disease, relieving acondition caused by the disease or disorder, or reducing the symptoms ofthe disease or disorder.

Chemical Description

The disclosure includes injectable nanoparticle neurosteroidformulations. The neurosteroid may be a compound of Formula I. Formula Iincludes allopregnanolone, ganaxolone, alphaxalone, alphadolone,hydroxydione, minaxolone, pregnanolone, acebrochol, ortetrahydrocorticosterone.

Ganaxolone (CAS Reg. No. 38398-32-2, 3α-hydroxy,3β-methyl-5α-pregnan-20-one) is a synthetic steroid with anti-convulsantactivity useful in treating epilepsy and other central nervous systemdisorders.

Ganaxolone has a relatively long half-life—approximately 20 hours inhuman plasma following oral administration (Nohria, V. and Giller, E.,Neurotherapeutics, (2007) 4(1): 102-105). Furthermore, ganaxolone has ashort T_(max), which means that therapeutic blood levels are reachedquickly. Thus initial bolus doses (loading doses) may not be required,which represents an advantage over other treatments. Ganaxolone isuseful for treating seizures in adult and pediatric epileptic patients.

Allopregnanolone (CAS Reg. No. 516-54-1, 3α,5α-tetrahydroprogesterone)is an endogenous progesterone derivative with anti-convulsant activity.

Allopregnanolone has a relatively short half-life, about 45 minutes inhuman plasma. In addition to its efficacy in treating seizures,allopregnanolone is being evaluated for use in treatingneurodegenerative diseases including Alzheimer's disease, Parkinson'sdisease, Huntington's disease, and amyotrophic lateral sclerosis and fortreating lysosomal storage disorders characterized by abnormalities incholesterol synthesis, such as Niemann Pick A, B, and C, Gaucherdisease, and Tay Sachs disease. (See U.S. Pat. No. 8,604,011, which ishereby incorporated by reference for its teachings regarding the use ofallopregnanolone for treating neurological disorders.)

Alphaxalone, also known as alfaxalone, (CAS Reg. No. 23930-19-0,3α-hydroxy-5α-pregnan-11, 20-dione) is a neurosteroid with an anestheticactivity. It is used as a general anaesthetic in veterinary practice.Anaesthetics are frequently administered in combination withanti-convulsants for the treatment of refractory seizures. An injectablenanoparticle neurosteroid dosage form containing alphaxalone alone or incombination with either ganaxolone or allopregnanolone is within thescope of this disclosure.

Alphadolone, also known as alfadolone, (CAS Reg. No. 14107-37-0, 3α,21-dihydroxy-5α-pregnan-11, 20-dione) is a neurosteroid with anaestheticproperties. Its salt, alfadolone acetate is used as a veterinaryanaesthetic in combination with alphaxalone.

Additional neurosteroids that may be used in the injectable nanoparticleneurosteroid formulation of this disclosure include formulations includehydroxydione (CAS Reg. No. 303-01-5,(5β)-21-hydroxypregnane-3,20-dione), minaxolone (CAS Reg. No.62571-87-3,2β,3α,5α,11α)-11-(dimethylamino)-2-ethoxy-3-hydroxypregnan-20-one),pregnanolone (CAS Reg. No, 128-20-1, (3α,5β)-d-hydroxypreganan-20-one),renanolone (CAS Reg. No, 565-99-1, 3α-hydroxy-5β-pregnan-11,20-dione),or tetrahydrocorticosterone (CAS Reg. No. 68-42-8,3α,5α-pregnan-20-dione).

Neurosteroid Nanoparticles

This disclosure is directed to injectable nanoparticle formulations,including formulations suitable for intravenous administration. Theneurosteroid nanoparticles contain a neurosteroid of Formula I, asurface stabilizer, and a surfactant. In certain embodiments theneurosteroid, may be ganaxolone or allopregnanolone.

This disclosure is also directed to neurosteroid nanoparticles aneurosteroid of Formula I, a surface stabilizer, and a surfactant.

The disclosure provides injectable neurosteroid nanoparticleformulations, including formulations containing nanoparticles comprisinga neurosteroid of Formula I, at least one surface stabilizer, such ashydroxyethyl starch, dextran, or povidone and a surfactant. In certainembodiments the nanoparticles comprise ganaxolone or allopregnanolone,hydroxyethyl starch, and a surfactant. Injectable neurosteroidnanoparticle formulations disclosed herein include formulations suitablefor intramuscular, intravenous, intraarterial, intraspinal, subcutaneousand intrathecal injection. Injectable formulations include parenteralformulations suitable for intravenous infusion.

Many neurosteroids are very poorly soluble in water and thus difficultto formulate as aqueous injectable dosage forms. For example, ganaxoloneis very poorly soluble in water (<0.001 mg/mL). The inventors have foundthat neurosteroids may be formulated as an aqueous injectable suspensionby preparing the neurosteroid as a nanoparticle, the nanoparticleparticle containing a polymeric surface stabilizer, such as eitherhydroxyethyl starch, dextran, or povidone, and an additional surfacestabilizer, and the additional surface stabilizer is a surfactant.

The injectable neurosteroid nanoparticle formulation includes a surfacestabilizer. In certain embodiment the surface stabilizer is a bloodreplacer; such as a blood volume expander. In certain embodiments thesurface stabilizer is either hydroxyethyl starch, dextran, or povidone.Hydroxyethyl starch is used as a blood volume expander in patientssuffering from severe blood loss. Grades of hydroxyethyl starch suitablefor use in the neurosteroid nanoparticles include 130/0.4 (CAS Reg. No.9005-27-0). In certain embodiments the surface stabilizer is dextran.Dextran is a single chain branched glucan having chains of varyinglengths. Like hydroxyethyl starch, dextran is also used as a bloodvolume expander. Dextrans are classified according to MW. Dextranshaving molecular weights from 40 kD to 75 kD have been used as bloodvolume expanders. Suitable dextrans for intravenous use include Dextran40, Dextran 60, Dextran 70, and Dextran 75. In certain embodiments thesurface stabilizer is a dextran having a molecular weight from about 40kD to about 75 kD. In certain embodiments the surface stabilizer isDextran 70. Povidone, also known as polyvinylpyrrolidone, is anotherapproved plasma expander. Povidone includes PLASDONE C-12 and C-17 fromAshland, Inc.

Other excipients useful as surface stabilizers for the injectableneurosteroid nanoparticle formulation include human serum albumin,hydrolyzed gelatin, polyoxyethylene castor oil, and polyoxyethylenehydrogenated castor oil. The injectable neurosteroid nanoparticleinjectable formulation includes a surfactant.

Surfactants include compounds such as lecithin (phosphatides), sorbitantrioleate and other sorbitan esters, polyoxyethylene sorbitan fatty acidesters (e.g., the commercially available TWEENS such as polyoxyethylenesorbitan monolaurate (TWEEN 20) and polyoxyethylene, sorbitan monooleate(TWEEN 80) (ICI Specialty Chemicals)); poloxamers (e.g., poloxamer 188PLURONIC F68 and poloxamer 338 (PLURONIC F108), which are blockcopolymers of ethylene oxide and propylene oxide), lecithin, sodiumcholesterol sulfate or other cholesterol salts, and bile salts, such assodium deoxycholate. Additional bile salts that may be used assurfactants include sodium cholate, sodium glycholate, salts ofdeoxycholic acid, salts of glycholic acid, salts of chenodeoxycholicacid, and salts of lithocholic acid.

The disclosure includes neurosteroid nanoparticles having a volumeweighted median diameter (D50) of from about 50 nm to about 2000 nm,about 50 nm about 500 nm, about 10 nm to about 350 nm, or having a D50of from about 50 nm to about 300 nm, or having a D50 of from about 100nm to about 250 nm, or having a D50 of about 150 nm to about 220 nm, orhaving a D50 of less than 2000 nm, less than 500 nm, of less than 350nm, less than 300 nm, less than 250 nm, or less than 200 nm. In oneaspect the neurosteroid nanoparticles have at least one of the followingproperties: (a) greater than 90% of the neurosteroid by weight is in theform of submicron particle having an effective size of about 50 nm toabout 250 inn; (b) at least about 20% of the neurosteroid by weight isin the form of an amorphous powder; (c) at least about 50% of theneurosteroid by weight is in the form of a crystalline powder of asingle polymorph; (d) at least about 50% of the neurosteroid is in theform of a semi-crystalline powder; (e) the neurosteroid is in the formof particles wherein at least about 50%, or at least 60%, or at least70%, or at least 80%, or at least 90% of the particles by weight have aneffective size less than 300 nm; (f) the neurosteroid is in the form ofparticles wherein at least about 50% of the particles by weight have aneffective size less than 250 nm; (g) the neurosteroid is in the form ofparticles having a D50 of about 50 nm to about 200 nm, wherein theparticle size distribution is described by a three-slice model in whicha certain percentage has an effective particle size by weight betweenabout 10 nm and about 100 nm, a certain percentage has an effectiveparticle size by weight between about 100 nm and about 200 nm, and acertain percentage has an effective particle size by weight above 200nm, and further wherein the three-slice model is identified as x %/y %/z%, respectively (e.g., 40%/30%/30%); (p) the neurosteroid has athree-slice distribution selected from the group 40%/30%/30%,50%/30%/20%, 60%/30%/10%, 40%/40%/20%, 50%/40%/10%, 70%/20%/10%50%/45%/5%, 70%/25%/5%, 60%/35%/5% 80%/15%/5%, 70%/30%/0%, 60%/40%/0%,90%/10%/0%, and 100%/0/0%; (h) the neurosteroid is in the form ofparticles, wherein standard deviation of the particle size distributiondivided by the volume-weighted mean diameter is less than about 30%,less than about 25%, less than about 20%, less than about 15%, or lessthan about 10%. In alternative embodiments, the neurosteroid in thecomposition has at least two of the aforementioned properties; at leastabout three of the aforementioned properties least about four of theaforementioned properties; or at least five of the aforementionedproperties.

The neurosteroid nanoparticles may be prepared by grinding. Grinding cantake place in any suitable grinding mill. Suitable mills include an airjet mill, a roller mill, a ball mill, an attritor mill, a vibratorymill, a planetary mill, a sand mill and a bead mill. A high energy mediamill is preferred when small particles are desired. The mill can containa rotating shaft.

The preferred proportions of the grinding media, neurosteroid, theoptional liquid dispersion medium, and dispersing, wetting or otherparticle stabilizing agents present in the grinding vessel can varywithin wide limits and depends, for example, the size and density of thegrinding media, the type of mill selected, the time of milling, etc. Theprocess can be carried out in a continuous, batch or semi-batch mode. Inhigh energy media mills, it can be desirable to till 80-95% of thevolume of the grinding chamber with grinding media. On the other hand,in roller mills, it frequently is desirable to leave the grinding vesselup to half filled with air, the remaining volume comprising the grindingmedia and the liquid dispersion media, if present. This permits acascading effect within the vessel on the rollers which permitsefficient grinding. However, when foaming is a problem during wetgrinding, the vessel can be completely filled with the liquid dispersionmedium or an anti-foaming agent may be added to the liquid dispersion.

The attrition time can vary widely and depends primarily upon the drug,mechanical means and residence conditions selected, the initial anddesired final particle size and so forth.

After attrition is completed, the grinding media is separated from themilled neurosteroid particulate product (in either a dry or liquiddispersion form) using conventional separation techniques, such as byfiltration, sieving through a mesh screen, and the like.

In one aspect, the grinding media comprises beads having a size rangingfrom 0.05-4 mm, preferably 0.1-0.4 mm. For example, high energy millingof neurosteroid with yttrium stabilized zirconium oxide 0.4 mm beads fora milling residence time of 25 minutes to 1.5 hours in recirculationmode at 2500 RPM. In another example, high energy milling ofneurosteroid with plastic beads (e.g. Purolite® Puromill 300) for amilling time of 400 minutes in recirculation mode at 4200 RPM. Inanother example, high energy milling of neurosteroid with 0.1 mmzirconium oxide balls for a milling residence time of 2 hours in batchmode. Additionally, the milling temperature should not exceed 50″ C asthe viscosity of the suspension may change dramatically. The millingconcentration is from about 1% to about 40% neurosteroid by weight. Inone embodiment, the concentration is 25% neurosteroid by weight. In oneembodiment, the milling media contains at least one agent to adjustviscosity so that the desired particles are suspended evenly, and awetting and/or dispersing agent to coat the initial neurosteroidsuspension so a uniform feed rate may be applied in continuous millingmode. In another embodiment, batch milling mode is utilized with amilling media containing at least one agent to adjust viscosity and/orprovide a wetting effect so that the neurosteroid is well dispersedamongst the grinding media.

Injectable Neurosteroid Nanoparticle Formulations

The disclosure provides injectable neurosteroid nanoparticleformulations containing the neurosteroid at a concentration of about0.25 mg/mL, about 0.5 mg/mL, about 1.0 mg/mL, about 1.5 mg/mL, about 2.0mg/mL, about 2.5 mg/mL, about 3.0 mg/mL, about 3.5 mg/mL, about 4.0mg/mL, about 4.5 mg/mL, about 5.0 mg/mL, about 5.5 mg/mL, about 6.0mg/mL, about 6.5 mg/mL, about 7.0 mg/mL, about 7.5 mg/mL, about 8.0mg/mL, about 8.5 mg/mL, about 9.0 mg/mL, about 10 mg/mL, about 11 mg/mL,about 12 mg/mL, about 13 mg/mL, or about 15 mg/mL. All ranges includingany two of the foregoing concentrations of neurosteroid as endpoints arealso included in the disclosure. For example, the disclosure includesneurosteroid nanoparticle formulations containing from about 0.5 mg/mLto about 15 mg/mL, about 1.0 mg/mL to about 10 mg/mL, about 2.0 mg/mL toabout 8.0 mg/mL, or about 4.0 mg/mL to about 8.0 mg/mL neurosteroid.

The nanoparticles will include neurosteroid and a surface stabilizer,such as either hydroxyethyl starch, povidone, or dextran, in a weight toweight ratio of neurosteroid to surface stabilizer is about 10:1 to0.5:1, or about 5:1 to about 0.5:1, or about 4:1 to about 1:1, or about3.5:1 to about 3:1, or about 3.3:1.

The disclosure includes embodiments in which the injectable neurosteroidnanoparticle formulation additionally comprises a buffer. In certainembodiments the buffer is a phosphate buffer. In certain embodiments thebuffer is phosphate buffered saline.

The injectable neurosteroid nanoparticle formulations may also includean acid or base buffer to adjust pH to desired levels. In someembodiments the desired pH is 2.5-11.0, 3.5-9.0, or 5.0-8.0, or 6.0-8.0,or 7.0-7.6, or about 7.4. Examples of acid buffers useful in theinjectable neurosteroid nanoparticle formulation include oxalic acid,maleic acid, fumaric acid, lactic acid, malic acid, tartaric acid,citric acid, benzoic acid, acetic acid, methanesulfonic acid, histidine,succinic acid, toluenesulfonic acid, benzenesulfonic acid,ethanesulfonic acid and the like. Acid salts of the above acids may beemployed as well. Examples of base buffers useful in the formulationinclude carbonic acid and bicarbonate systems such as sodium carbonateand sodium bicarbonate, and phosphate buffer systems, such as sodiummonohydrogen phosphate and sodium dihydrogen phosphate. Theconcentration of each component of a phosphate buffer system will befrom about 10 mM to about 200 mM, or from about 20 mM to about 150 mM,or from about 50 mM to about 100 mM.

The disclosure includes embodiments in which the pH of the neurosteroidnanoparticle formulation is about 7.4.

The formulation may contain electrolytes, such as sodium or potassium.The disclosure includes embodiments in which the formulation is fromabout 0.5% to about 1.5% sodium chloride (saline).

The formulation may contain tonicity adjusting agents so that it isisotonic with human plasma. Examples of tonicity adjusting agents usefulin the formulation include, but are not limited to, dextrose, mannitol,sodium chloride, or glycerin. In certain embodiments the tonicity agentis 0.9% sodium chloride.

The injectable neurosteroid nanoparticle formulations may contain anypharmaceutically acceptable excipient compatible with the neurosteroidand capable of providing the desired pharmacological release profile forthe dosage form. Excipients include, for example, suspending agents,surfactants, solubilizers, stabilizers, lubricants, wetting agents,anti-foaming agent, diluents, and the like. Pharmaceutically acceptableexcipients may comprise, but are not limited to, acacia, gelatin,colloidal silicon dioxide, calcium glycerophosphate, calcium lactate,maltodextrin, glycerin, magnesium silicate, polyvinylpyrrolidone (PVP),cholesterol, cholesterol esters, sodium caseinate, soy lecithin,taurocholic acid, phosphotidylcholine, sodium chloride, tricalciumphosphate, dipotassium phosphate, cellulose and cellulose conjugates,sugars sodium stearoyl lactylate, carrageenan, monoglyceride,diglyceride, pregelatinized starch, and the like.

Suitable antifoaming agents include dimethicone, myristic acid, palmiticacid, and simethicone.

The injectable neurosteroid nanoparticle formulation may also contain anon-aqueous diluent such as ethanol, one or more polyol (e.g. glycerol,propylene glycol), an oil carrier, or any combination of the foregoing.

H The injectable neurosteroid nanoparticle formulation may additionallycomprise a preservative. The preservative may be used to inhibitbacterial growth or prevent deterioration of the active agent.Preservatives suitable for parenteral formulations include ascorbicacid, acetylcysteine, benzalkonium chloride, benzethonium chloride,benzoic acid, benzyl alcohol, chlorbutanol, chlorhexidene, m-cresol,2-ethoxyethanol, human serum albumin, monothioglycerol, parabens(methyl, ethyl, propyl, butyl, and combinations), phenol,phenylmercurate salts (acetate, borate nitrate), sorbic acid, sulfurousacid salts (bisulfite and metabisulfite), and thimerosal. In certainembodiments the preservative is an antioxidants such ascorbic acid,glutathione, or an amino acid. Amino acids useful as antioxidantsinclude methionine, cysteine, and L-arginine.

Lyophilized Neurosteroid Nanoparticle Formulations

The disclosure includes lyophilized forms of all formulations disclosedherein.

The injectable neurosteroid nanoparticle formulations provided in thisdisclosure are aqueous formulations or powder formulations includinglyophilized forms, which may be readily resuspended in water to providean injectable formulation. The disclosure includes embodiments in whichthe lyophilized neurosteroid powder comprises the neurosteroid, asurface stabilizer such as either hydroxyethyl starch or dextran, and asurfactant, wherein the injectable formulation is about 0.5% to about40% neurosteroid, about 0.5% to about 20% neurosteroid, about 0.5% toabout 10% neurosteroid, about 0.5% to about 2.0%, or about 1.0% to about1.5% weight neurosteroid.

The disclosure provides injectable neurosteroid nanoparticleformulations containing neurosteroid nanoparticles containingneurosteroid and an excipient, such as hydroxy ethyl starch or dextran,and optionally a surfactant. In certain embodiments the neurosteroidnanoparticle formulation is a lyophilized form that is dissolved inwater or an aqueous solution prior to administration.

The lyophilized form may additionally include an antifoaming agent, abuffer (or pH adjuster), a cryoprotectant, a hulking agent, a tonicityadjuster, or a combination of any of the foregoing.

Bulking agents are useful for lyophilized formulation in which a lowconcentration of the active ingredient, or in the present case, in whicha low concentration of the inclusion complex, is present. Bulking agentsinclude mannitol, lactose, sucrose, trehalose, sorbitol, glucose,rafinose, glycine, histidine, polyethylene glycol (PEG), and polyvinylpyrrolidone (PVP).

The removal of the hydration shell from an active agent duringlyophilization can be destabilizing. In certain embodiments thelyophilized form contains a stabilizer which serves as a cryoprotectant.Stabilizers include agents which maintain a desirable attribute of theformulation over a time interval including but not limited tomechanical, chemical and temperature stressing that can be tested in alaboratory setting. Such attributes include stable particle size orhomogeneity resulting in concentrations consistent with the labeledpotency and maintaining purity.

Suitable cryoprotectant stabilizers include sugars such as sucrose,trehalose, glucose, rafinose, lactose, mannitol, sorbitol, histidine,polyethylene glycol (PEG), and polyvinyl pyrrolidone and sodiumchloride.

Ebeam Sterilized Nanoparticulate Formulations

Electron beam sterilization (ebeam) is a process using beta radiation,usually of high energy, to effect sterilization of a sample.Surprisingly, it has been determined that the injectable nanoparticleneurosteroid formulations of this disclosure can be sterilized withebeam radiation without affecting particle size, impurity levels orviscosity. Lyophilized powders of the injectable nanoparticleneurosteroid formulations may also be sterilized with ebeam radiationwithout adverse effects.

Additional embodiments of the disclosure include injectable nanoparticleneurosteroid formulations sterilized with ebeam irradiation. Lyophilizedpowders or other dry forms of such formulations are also included inthis disclosure. The injectable nanoparticle neurosteroid formulationsof this disclosure can be subjected to ebeam irradiation, preferably atambient temperature. This temperature remains relatively constant duringirradiation.

The ebeam radiation is applied in an amount sufficient to destroysubstantially all of the microbial contamination in the dispersion. Thetotal amount of ebeam radiation that dispersion is exposed to has beenexperimentally verified to: (1) show only a modest increase in particlesize on storage following exposure to ebeam irradiation, (2) maintainthe integrity of the nanoparticulate active agent, and (3) to showacceptable impurity concentrations following ebeam irradiation. Theapplication of the ebeam radiation does not significantly degrade theneurosteroid or reduce its efficacy. The present disclosure enablesproducts which meet cGMP requirements for sterile products withoutharming the neurosteroid nanoparticles.

In certain embodiments the ebeam radiation is applied in a cumulativeamount of 25 kGray. Generally, the ebeam radiation will normally beapplied in a range of 5 kGray to 50 kGray, 5 kGray to 40 kGray, 10 kGrayto 30 kGray, 5 to 15 kGray, or 5 to 10 kGray. Multiple doses ofradiation can be utilized to achieve a desired cumulative radiationdosage.

The microbial contamination which is to be destroyed is generally thatof bacterial contamination and mycoplasma contamination.

Surprisingly, following sterilization the injectable neurosteroidnanoparticle formulations exhibit unexpected overall stability,maintaining the pre-sterilized physical and chemical properties, whilemeeting cGMP requirements for sterility. The overall stability of theebeam irradiated dispersions of nanoparticulate neurosteroid wasmeasured in terms of neurosteroid nanoparticle particle size, content ofdegradation products, and viscosity. It is particularly unexpected thatebeam irradiation of the injectable neurosteroid nanoparticleformulations does not significantly alter the particle size of theneurosteroid nanoparticles. This is significant because if thesterilized product formed aggregates or large crystals, the dispersionwould no longer be useful as an injectable formulation. Other means ofsterilization including heat sterilization were found to alter theneurosteroid nanoparticle particle size.

Methods of Treatment

The disclosure includes methods of treating status epilepticus,refractory status epilepticus, super-refractory status epilepticus,PCDH19 female pediatric epilepsy, and other seizure disorders comprisingadministering an effective amount of the neurosteroid nanoparticleinjectable formulation to a patient suffering from any of these seizuredisorders.

Seizure disorders that may be treated with the neurosteroid nanoparticleinjectable formulation include status epilepticus, e.g., convulsivestatus epilepticus, e.g., early status epilepticus, established statusepilepticus, refractory status epilepticus, super-refractory statusepilepticus, e.g., super-refractory generalized status epilepticus;non-convulsive status epilepticus, e.g., generalized status epilepticus,complex partial status epilepticus; a seizure, e.g., acute repetitiveseizures, cluster seizures, infantile spasms, Lennox-Gastaut syndrome,West syndrome, PCDH19 female pediatric epilepsy, and catamenialepilepsy.

The neurosteroid nanoparticle injectable formulation may also be used totreat provoked seizures such as seizures resulting from low blood sugar,electrolyte imbalance, high fever, brain infection (such as braininfections due to encephalitis, malaria, meningitis, toxoplasmosis, oramoebic infection), adverse reaction to prescription drugs, or alcoholor drug overdose.

The disclosure also includes methods of using neurosteroid nanoparticleinjectable formulation to treat traumatic brain injury and strokecomprising administering an effective amount of the formulation to apatient suffering from recent traumatic brain injury or a recent stroke.

The disclosure further includes methods of treating seizures arisingfrom neurodegenerative disorders. Such neurodegenerative disordersinclude Parkinson's disease, Alzheimer's disease, Amyotrophic LateralSclerosis, and Huntington's disease. The disclosure includes methods oftreating seizure arising from inflammatory disorders, such as multiplesclerosis. The disclosure includes methods of treating seizure disordersarising from lysosomal storage disorders including Neimann-Pick-C, TaySachs, Batten, Sandhoff, and Gaucher disease.

Methods of treatment include treating a patient suffering from seizures,traumatic brain injury, or stroke by administering a single injection(bolus dose) of a neurosteroid nanoparticle injectable formulation. Thesingle injection may be administered intramuscularly or intravenously.The dose of the single injection may be from about 0.5 mg/kg to about 20mg/kg, from about 2 mg/kg to about 15 mg/kg, from about 2 mg/kg to about10 mg/kg, or about 2 mg/kg to about 8 mg/kg. Methods of treatment alsoinclude administering multiple injections of the neurosteroidnanoparticle injectable formulation over a period of 1 to 10 days. Theinjections may be given at intervals of 1 to 24 hours. Dosing schedulesin which the injectable neurosteroid nanoparticle formulation isinjected every 1 hour, 2 hours, 4 hours. 6 hours, 8 hours. 12 hours, or24 hours are included herein. Dosing schedules in which the neurosteroidnanoparticle injectable formulation is injected for 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 days are included herein.

Methods of treatment include treating a patient suffering from seizures,traumatic brain injury, or stroke by administering one or more bolusdoses over a period of 1 to 10 days as described in the precedingparagraph of a neurosteroid nanoparticle injectable formulation followedby an intravenous infusion of the neurosteroid nanoparticle injectableformulation. In certain embodiments the bolus dose is administered overa period of about 1 to about 30, about 1 to about 15, about 1 to about10, or about 1 to about 5, or about 5 minutes followed by commencementof the intravenous infusion within 1, 2, 3, 4, or 5 hours.

In some embodiments, neurosteroid nanoparticle injectable formulation isadministered as an intravenous infusion dose, either with or without aprevious bolus dose for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutivedays. The infusion dose may be administered at a rate of 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 mg/kg hr or in a range of about 1 mg/kg/hr to about 10mg/kg/hr or 2 mg/kg/fir to about 8 mg/kg/hrs.

In some embodiments the infusion dose (whether administered with orwithout the bolus dose) is followed by a first step down dosage, andoptionally a second step down dosage, an optionally a third step downinfusion dosage. In some embodiments, the first step dose is 95%, 90%,85%, 80%, 75%, 70%, 65%, 60%. 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%,15%, 10%, or 5% of the infusion dose. In some embodiments, the firststep dose is between 95-50%, 75-50%, 85-50%, 90-50%, 80-50%, or 75-100%of the infusion dose. In an embodiment, the first step dose is 75% ofthe infusion dose. In some embodiments, the second step dose is 95%,90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%,20%. 15%, 10%, or 5% of the first step down dose. In some embodiments,the second step dose is between 95-30%, 75-30%, 85-30%, 60-30%, 70-30%,50-30%, or 50-40% of the first step down dose. In an embodiment, thesecond step dose is 50% of the infusion dose. In some embodiments, thethird step dose is 95%, 90%, 85%. 80%, 75%, 70%, 65%, 60%, 55%, 50%,45%, 40%. 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the second infusiondose. In some embodiments, the third step dose is between 50-5%, 40-5%,30-5%, 25-5%, 25-10% 25-20%, or 25-40% of the second step down dose. Inan embodiment, the third step down dose is 25% of the infusion dose.

The disclosure includes methods of treating a seizure disorder whereinthe seizure disorder is status epilepticus, refractory statusepilepticus, super refractory status epilepticus, or PCDH19 femalepediatric epilepsy comprising administering an effective amount of theneurosteroid nanoparticle injectable formulation to a patient.

The disclosure includes methods of treating a seizure disorder, stroke,or traumatic brain injury, comprising administering an effective amountof the neurosteroid nanoparticle injectable formulation to a patientwherein the amount of neurosteroid administered is from about 1 mg/kg toabout 200 mg/kg.

In certain embodiments the neurosteroid nanoparticle injectableformulation is administered intramuscularly or intravenously.

The disclosure includes embodiments in which the neurosteroidnanoparticle injectable formulation is administered as a single bolusdose of the neurosteroid formulation to the patient. In certainembodiments the single bolus dose provides a sufficient amount ofneurosteroid to provide a plasma C_(max) of neurosteroid of about 100ng/mL to about 1000 ng/mL in the patient.

The disclosure includes embodiments in which the neurosteroidnanoparticle injectable formulation is administered as a bolus dose andthe bolus dose provides a sufficient amount of neurosteroid to provide aplasma C_(max) of neurosteroid of about 100 ng/mL to about 800 ng/mL inthe patient.

The disclosure includes embodiments in which the neurosteroidnanoparticle formulation is administered as a bolus dose and the bolusdose is administered in less than 10 minutes and the C_(max) occurswithin 1 hour of completion of administration.

The disclosure includes embodiments in which the neurosteroidnanoparticle formulation is administered as a single bolus dose and thesingle bolus dose comprises from about 0.5 mg/kg to about 20 mg/kgneurosteroid. Or, optionally the single bolus dose comprises from about2 mg/kg to about 15 mg/kg neurosteroid, or about 4 mg/kg to about 10mg/kg neurosteroid, or from about 1 mg/kg to about 30 mg/kgneurosteroid.

The disclosure includes embodiments in which multiple bolus doses of theneurosteroid nanoparticle formulation are administered to the patient.In certain embodiments the multiple bolus doses are given over 1 to 10days at intervals of 1 to 24 hours. In certain embodiments each bolusdose provides a sufficient amount of neurosteroid to produce a plasmaC_(max) of neurosteroid of about 100 ng/mL to about 8000 ng/mL in thepatient. In certain embodiments the interval between bolus doses is fromabout 10 to about 24 hours and once an initial C_(max) is reached theplasma concentration of neurosteroid is not below 100 ng/mL at any timebetween bolus doses. In certain embodiments the interval between bolusdoses is 20 to 24 hours and once an initial C_(max) is reached and theconcentration of neurosteroid in the patient's plasma does not fallbelow 25% of the initial C_(max). In certain embodiment each bolus dosecomprises about 1 mg/kg to about 20 mg/kg neurosteroid. Or, optionallythe single bolus dose comprises from about 2 mg/kg to about 15 mg/kgneurosteroid, or about 4 mg/kg to about 10 mg/kg neurosteroid, or fromabout 1 mg/kg to about 30 mg/kg neurosteroid.

In certain embodiments the method comprises administering an infusion ofthe neurosteroid nanoparticle formulation to the patient, with orwithout an initial bolus dose. In certain embodiments the infusion isadministered for 1 to 10 consecutive days at a rate of 1 to 10 mg/kg/hrwithout an initial bolus dose.

In certain embodiments the method comprises administering an initialbolus dose of the neurosteroid nanoparticle injectable formulationcomprising from about 1 mg/kg to about 20 mg/kg neurosteroid, followedwithin 24 hours by administration of an infusion of the neurosteroidformulation for 1 to 10 consecutive days at a rate of 1 to 10 mg/kg/hr.

In certain embodiments the method comprises administering an initialbolus dose of the neurosteroid nanoparticle injectable formulationfollowed by an infusion dose, wherein the initial bolus dose provides asufficient amount of neurosteroid to provide an initial plasma C_(max)of neurosteroid of about 100 ng/mL to about 1000 ng/mL in the patientand the concentration of neurosteroid in the patient's plasma does notfall below 25% of the initial C_(max) until after the subsequentinfusion dosing is concluded.

In certain embodiments the method comprises administering an initialbolus dose of the neurosteroid nanoparticle injectable formulation,wherein the initial bolus dose provides a sufficient amount ofneurosteroid to provide an initial plasma C_(max) of neurosteroid ofabout 100 ng/mL to about 8000 ng/mL in the patient, the patient is thenadministered an infusion of the neurosteroid formulation at a constantdose sufficient to provide a concentration of neurosteroid in thepatient's plasma of at least 40% of C_(max), followed by an infusion ofneurosteroid at a gradually reducing dose so that the concentration ofneurosteroid in the patient's plasma is less than 20% of C_(max) whenthe infusion is concluded.

Combination Treatment

The disclosure includes embodiments in which the neurosteroid is theonly active agent and embodiments in which the neurosteroid isadministered in combination with one or more additional active agents.When used in combination with an additional active agent theneurosteroid and the additional active agent may be combined in the sameformulation or may be administered separately. The neurosteroid may beadministered while the additional active agent is being administered(concurrent administration) or may be administered before or after theadditional active agent is administered (sequential administration).

The disclosure includes embodiments in which the additional active agentis an anti-convulsant. Anticonvulsants include GABA_(A) receptormodulators, sodium channel blocker, GAT-1 GABA transporter modulators,GABA transaminase modulators, voltage-gated calcium channel blockers,and peroxisome proliferator-activated alpha modulators.

The disclosure includes embodiments in which the patient is given ananesthetic or sedative in combination with a neurosteroid. Theanesthetic or sedative may be administered at a concentration sufficientto cause the patient to lose consciousness, such as a concentrationsufficient to medically induce coma or a concentration effective toinduce general anesthesia. Or the anesthetic or sedative may be given ata lower dose effective for sedation, but not sufficient to induce a lossof consciousness.

A medically induced coma occurs when a patient is administered a dose ofan anesthetic, such as propofol, pentobarbital or thiopental, to cause atemporary coma or a deep state of unconsciousness. General anesthesia isa treatment with certain medications to cause unconsciousness sufficientto be unaware of pain during surgery. Drugs used for medically inducedcoma or general anesthesia include inhalational anesthetics andintravenous anesthetics which include barbiturate and non-barbiturateanesthetics.

Inhalational anesthetics include desflurane, enflurane, ethyl chloride,halothane, isoflurane, methoxyflurane, sevoflurane, andtrichloroethylene.

Intravenous, non-barbiturate anesthetics include atracurium,cisatracurium, etodimidate, ketamine, propofol, and rocuronium,

Barbiturates include amobarbital, methohexital, pentobarbital,phenobarbital, secobarbital, thiamylal, and thiopental.

Benzodiazepines are used both as anticonvulsants and anesthetics.Benzodiazepines useful as anaesthetics include diazepam, flunitrazepam,lorazepam, and midazolam.

The disclosure includes administering propofol to induce anesthesia incombination with a neurosteroid. Propofol is administered at a doserange or dosage range of 0.5-50 mg/kg. Anesthesia is induced with aninitial bolus of 10-50 mg/kg followed by additional intermittent bolusesor 10-50 mg/kg to maintain anesthesia. Anesthesia may also be maintainedby an infusion of 3-18 mg/kg/min propofol.

The disclosure includes administering pentobarbital sodium byintravenous or intramuscular injection to induce anesthesia incombination with a neurosteroid. Pentobarbital may be administered toadults as a single 100-500 mg, or 100-200 mg intramuscular orintravenous injection, or to pediatric patients as a single 2 to 6 mg/kgIM or IV injection. Pentobarbital may be administered at a high dose toinduce coma in a status epilepticus patient and a neurosteroid may thenbe given in combination with the pentobarbital to treat refractoryseizures. Pentobarbital doses used to induce coma include, a loadingdose of 5 to 15 mg/kg or 10 to 35 mg/kg, given over 1-2 hours followedby a maintenance dose of 1 mg/kg/hr to 5 mg/kg/hr for 12 to 48 hours andtapering by 0.25 to 0.5 mg/kg/hr every 12 hours once seizures havestopped.

The disclosure includes administering thiopental sodium in combinationwith a neurosteroid. Thiopental can be administered as a 3 to 5 mg/kgbolus followed by additional boluses of 1 to 2 mg/kg every 3 to 5minutes until seizures have stopped, to a maximum total dose of 10mg/kg. After the 10 mg/kg maximum bolus dose of thiopental has beenreached, thiopental can be infused at 3 to 5 mg/kg/hr.

The disclosure includes administering midazolam in combination with aneurosteroid. Midazolam can be administered as a 0.5 mg/kg to 5 mg/kgloading dose, followed by a 1 to 5 microgram/kg/hour infusion.

In each embodiment in which an additional active agent is administeredto induce coma, anesthesia, or sedation, a neurosteroid is administeredas a neurosteroid nanoparticle injectable formulation and isadministered simultaneously or sequentially with the additional activeagent and is administered according to any of the dosing schedules seforth herein for neurosteroid administration.

The neurosteroid nanoparticle injectable formulation of this disclosuremay be administered with another anticonvulsant agent. Anticonvulsantsinclude a number of drug classes and overlap to a certain extent withthe coma-inducing, anesthetic, and sedative drugs that may be used incombination with a neurosteroid. Anticonvulsants that may be used incombination with the neurosteroid nanoparticle injectable formulation ofthis disclosure include aldehydes, such as paraldehyde; aromatic allylicalcohols, such as stiripentol; barbiturates, including those listedabove, as well as methylphenobarbital and barbexaclone; benzodiazepinesinclude alprazolam, bretazenil, bromazepam, brotizolam,chlordiazepoxide, cinolazepam, clonazepam, chlorazepate, clobazam,clotiazepam, cloxazolam, delorazepam, diazepam, estazolam, etizolam,ethyl loflazepate, flunitrazepam, flurazepam, flutoprazepam, halazepam,ketazolam, loprazolam, lorazepam, lormetazepam, medazepam, midazolam,nimetazepam, nitrazepam, nordazepam, oxazepam, phenenazepam, pinazepam,prazepam, premazepam, pyrazolam, quazepam, temazepam, tatrazepam, andtriazolam; bromides, such as potassium bromide; carboxamides, suchcarbamazepine, oxcarbazepine, and eslicarbazepine acetate; fatty acids,such as valproic acid, sodium valproate and divalproex sodium; fructosederivatives, such as topiramate; GABA analogs such as gabapentin andpregabalin, hydantoins, such as ethotoin, phenytoin, mephenytoin, andfosphenytoin; other neurosteroids, such as allopregnanolone,oxasolidinediones, such as paramethadione, trimethadione, and ethadione,propionates such as beclamide; pyrimidinediones such as primidone,pyrrolidines such as brivaracetam, levetiracetam, and seletracetam,succinimides, such as ethosuximide, pensuximide, and mesuximide;sulfonamides such as acetazoloamide, sultiame, methazolamide, andzonisamide; triazines such as lamotrigine, ureas such as pheneturide andphenacemide; NMDA antagonists, such as felbamate, and valproylamidessuch as valpromide and valnoctamide; and perampanel.

Specific Embodiments

The disclosure provides the following specific embodiments that arefurther illustrated by the examples that follow.

(1) An injectable neurosteroid formulation comprising nanoparticleshaving a D50 of less than 2000 nm, the nanoparticles comprising

a) a neurosteroid of the Formula I:

or a pharmaceutically acceptable salt thereof, wherein:

X is O, S, or NR¹⁰;

R¹ is hydrogen, hydroxyl, optionally substituted alkyl, optionallysubstituted heteroalkyl, optionally substituted aryl, or optionallysubstituted arylalkyl;

R⁴ is hydrogen, hydroxyl, oxo, optionally substituted alkyl, oroptionally substituted heteroalkyl,

R², R³, R⁵, R⁶, and R⁷ are each independently hydrogen, hydroxyl,halogen, optionally substituted alkyl, or optionally substitutedheteroalkyl;

R⁸ is hydrogen or alkyl and R⁹ is hydroxyl; or

R⁸ and R⁹ are taken together to form an oxo group;

R¹⁰ is hydrogen, hydroxyl, optionally substituted alkyl, optionallysubstituted heteroalkyl, optionally substituted aryl, or optionallysubstituted arylalkyl where

each alkyl is a C₁-C₁₀alkyl, C₃-C₆cycloalkyl,(C₃-C₆cycloalkyl)C₁-C₄alkyl, and optionally contains a single bondreplaced by a double or triple bond;

each heteroalkyl group is an alkyl group in which one or more methylgroup is replaced by an independently chosen —O—, —S—, —N(R¹⁰)—, —S(═O)—or —S(═O)₂—, where R¹⁰ is hydrogen, alkyl, or alkyl in which one or moremethylene group is replaced by —O—, —S—, —NH, or —N-alkyl; and (b) atleast one surface stabilizer.

(2) The injectable neurosteroid formulation of Specific Embodiment 1,wherein

X is O;

R¹ is C₁-C₂alkyl optionally substituted with hydroxyl;

R² and R⁵ are methyl;

R³ and R⁶ are hydrogen;

R⁴ is hydrogen, C₁-C₂alkyl, mono- or di-C₁-C₂alkylamino, or oxo;

R⁷ is hydrogen, C₁-C₂alkyl, or C₁-C₂alkoxy; and

R⁸ is hydrogen or methyl and R⁹ is hydroxyl; or R⁸ and R⁹ are takentogether to form an oxo group.

(3) The injectable neurosteroid formulation of Specific Embodiment 2,wherein R⁴ is hydrogen or oxo; and R⁸ is hydrogen or methyl and R⁹ ishydroxyl.

(4) The injectable neurosteroid formulation of any one of SpecificEmbodiments 1 to 3, wherein the formulation is an intravenousformulation. The injectable neurosteroid formulation of any one ofSpecific Embodiments 1 to 3, wherein the formulation is an intramuscularformulation. The injectable neurosteroid formulation of any one ofSpecific Embodiments 1 to 3, wherein the formulation is a subcutaneousformulation.

(5) The injectable neurosteroid formulation of Specific Embodiments 1 to4, wherein the neurosteroid is allopregnanolone, ganaxolone,alphaxalone, alphadolone, hydroxydione, minaxolone, pregnanolone, ortetrahydrocorticosterone.

(6) The injectable neurosteroid formulation of Specific Embodiment 5,wherein the neurosteroid is ganaxolone or allopregnanolone.

(7) The injectable neurosteroid formulation of Specific Embodiment 6,wherein the neurosteroid is ganaxolone.

(8) The injectable neurosteroid formulation of Specific Embodiment 6,wherein the neurosteroid is allopregnanolone.

(9) The injectable neurosteroid formulation of any one of SpecificEmbodiments 1 to 8 wherein the nanoparticles have a D50 of less than 500nm.

(10) The injectable neurosteroid formulation of Specific Embodiment 9,wherein the nanoparticles have a D90 of less than 500 nm.

(11) The injectable neurosteroid formulation of Specific Embodiment 9wherein the nanoparticles have a D50 of 10 nm to 300 nm.

(12) The injectable neurosteroid formulation of any one of SpecificEmbodiments 1 to 11, wherein the at least one surface stabilizer is apolymeric surface stabilizer.

(13) The injectable neurosteroid formulation of Specific Embodiment 12,wherein the polymeric surface stabilizer is hydroxyethyl starch,dextran, povidone, or a mixture of any of the foregoing.

(14) The injectable neurosteroid formulation of Specific Embodiment 13,wherein the surface stabilizer is hydroxyethyl starch. (Such ashydroxyethyl starch 130/0.4)

(15) The injectable neurosteroid formulation of Specific Embodiment 13,wherein the surface stabilizer is dextran having an average molecularweight of 40 kD to 75 kD.

(16). The injectable neurosteroid formulation of Specific Embodiment 15,wherein the dextran is Dextran 70.

(17) The injectable neurosteroid formulation of Specific Embodiment 13,wherein the surface stabilizer is povidone. (Such as plasdone C-12 orplasdone C-17)

(18). The injectable neurosteroid formulation of any one of SpecificEmbodiments 1 to 17, wherein the formulation comprises an additionalsurface stabilizer and the additional surface stabilizer is an ionic ornonionic surfactant.

(19) The injectable neurosteroid formulation of Specific Embodiment 18,wherein the surfactant is sodium cholate, sodium deoxycholate, sodiumcholesterol sulfate, or a mixture of any of the foregoing.

(20). The injectable neurosteroid formulation of Specific Embodiment 19,wherein the surfactant is sodium deoxycholate.

(21) The injectable neurosteroid formulation of any one of SpecificEmbodiments 1 to 20, wherein the formulation additionally comprises anantifoaming agent.

(22) The injectable neurosteroid formulation of Specific Embodiment 21,wherein the antifoaming agent is simethicone.

(23) The injectable neurosteroid formulation of any one of SpecificEmbodiments 1 to 22 additionally comprising a cryoprotectant.

(24) The injectable neurosteroid formulation of Specific Embodiment 23,wherein the cryoprotectant is sucrose, dextrose, lactose, D-sorbitol, ora mixture of any of the foregoing.

(25) The injectable neurosteroid formulation of Specific Embodiment 24,wherein the cryoprotectant is sucrose.

(26) The injectable neurosteroid formulation of any one of SpecificEmbodiments 1 to 25, wherein the formulation additionally comprises 0.5%to 1.5% sodium chloride (weight percent).

(27) The injectable neurosteroid formulation of Specific Embodiment 27,wherein the formulation comprises about 0.9% sodium chloride.

(28) The injectable neurosteroid formulation of any one of SpecificEmbodiments 1 to 27, additionally comprising a buffer.

(29) The injectable neurosteroid formulation of Specific Embodiment 28,wherein the buffer is a phosphate buffer.

(30) The injectable neurosteroid formulation of Specific Embodiment 29,wherein the buffer is phosphate buffered saline.

(31) The injectable neurosteroid formulation of any one of SpecificEmbodiments 1 to 30, additionally comprising a preservative.

(32) The injectable neurosteroid formulation of Specific Embodiment 31,wherein the preservative is benzyl alcohol, chlorbutanol,2-ethoxyethanol, parabens (including methyl, ethyl, propyl, butyl, andcombinations), benzoic acid, sorbic acid, chlorhexidene, phenol,3-cresol, thimerosal, a phenylmercurate salt, or a mixture of any of theforegoing.

(33) The injectable neurosteroid formulation of any one of the foregoingembodiments, wherein

the neurosteroid is ganaxolone or allopregnanolone,

the at least one surface stabilizer is a polymeric surface stabilizerselected from hydroxyethyl starch, dextran, povidone, or a mixture ofthe foregoing, and

the formulation comprises and additional surface stabilizer and theadditional surface stabilizer is a surfactant chosen from sodiumdeoxycholate or sodium cholesterol sulfate and the (wt:wt) ratio of theneurosteroid to the surface stabilizer is about 10:1 to about 1:1. (34)The injectable neurosteroid formulation of Specific Embodiment 33wherein the (wt:wt) ratio of the neurosteroid to the polymeric surfacestabilizer is about 4:1 to about 3:1.

(35) The injectable neurosteroid formulation of Specific Embodiment 34wherein the (wt:wt) ratio of the neurosteroid to the polymeric surfacestabilizer is about 3.3:1.

(36) The injectable neurosteroid formulation of any one of SpecificEmbodiment 1 to 35, wherein the ratio of neurosteroid to surfactant(w:w) is about 10:1.5 to about 10:0.1.

(37) The injectable neurosteroid formulation of any one of SpecificEmbodiments 23 to 36, wherein the ratio (w:w) of neurosteroid tocryoprotectant is 4:1 to 1:4.

(38) The injectable neurosteroid formulation of any one of SpecificEmbodiments claims 1 to 37, wherein the formulation is in the form of alyophilized powder.

(39) The injectable neurosteroid formulation any one of SpecificEmbodiments 1 to 37, wherein the formulation is an aqueous suspensionand the neurosteroid concentration is about 0.1 mg/mL to about 300mg/mL.

(40) The injectable neurosteroid formulation of any one of SpecificEmbodiments 1 to 37 or 39, wherein the weight percent of neurosteroid isfrom about 0.1% to about 30% and the neurosteroid is ganaxolone orallopregnanolone.

(41) The injectable neurosteroid formulation of Specific Embodiment 40,wherein the weight percent of ganaxolone or allopregnanolone is fromabout 0.5% to about 2.0%.

(42) The injectable neurosteroid formulation of any one of SpecificEmbodiments 23 to 42, wherein the weight percent of cryoprotectant inthe formulation is from about 5% to about 60%.

(43) The injectable neurosteroid formulation of Specific Embodiment 42,wherein the weight percent of cryoprotectant in the formulation is fromabout 10% to about 40%.

(44). The injectable neurosteroid formulation of any one of SpecificEmbodiments 1 to 43, having a pH in the range of approximately 2.5-11.0.

(45) The injectable neurosteroid formulation of Specific Embodiment 44,having a pH of about 7.0 to about 7.6.

(46) The injectable neurosteroid formulation of Specific Embodiment 1,wherein the formulation is an aqueous formulation comprising

(a) nanoparticles having a D50 of less than 500 nm, the nanoparticlescomprising ganaxolone, wherein the weight percent of the ganaxolone is 1to 10%;

(h) a polymeric surface stabilizer is hydroxyethyl starch, dextran,povidone, or a mixture of any of the foregoing, wherein the weightpercent of the polymeric surface stabilizer is 2 to 20%

(c) an additional surface stabilizer wherein the additional surfacestabilizer is an ionic or nonionic surfactant selected sodium cholate,sodium deoxycholate, sodium cholesterol sulfate, wherein the weightpercent surfactant is 0.1% to 2.0%, and

(d) an antifoaming agent.

(47) The injectable neurosteroid formulation of Specific Embodiment 1wherein the formulation is an aqueous formulation comprising

(a) nanoparticles having a D50 of less than 500 nm, the nanoparticlescomprising ganaxolone, wherein the weight percent of the ganaxolone isabout 5%;

(b) a polymeric surface stabilizer selected from hydroxyethyl starch130/0.4 or plasdone C-12, wherein the weight percent of the polymericsurface stabilizer is about 10%;

(c) an additional surface stabilizer wherein the additional surfacestabilizer is sodium deoxycholate, wherein the weight percent of sodiumdeoxycholate is about 0.75% and

(d) simethicone, wherein the weight percent of simethicone is 0.009%.(48) A method for sterilizing the injectable neurosteroid nanoparticleformulation of any one of Specific Embodiments 1 to 47, comprisingsubjecting the formulation to ebeam radiation, wherein the methodproduces a sterilized neurosteroid nanoparticle formulation containing adegradant concentration of not more than 0.2% w/w of neurosteroid.

(49) The injectable neurosteroid formulation of any one of SpecificEmbodiments 1 to 47, wherein the formulation has been sterilized byebeam irradiation and wherein the formulation contains a degradantconcentration of not more than 0.2% w/w of the neurosteroid.

(50) The injectable neurosteroid formulation of Specific Embodiment 49,wherein the ebeam irradiation is a cumulative dose of about 25 kGray.

(51) The injectable neurosteroid formulation of Specific Embodiment 50,wherein the ebeam irradiation is a dose selected from 5 kGray to 50kGray, 5 kGray to 30 kGray, 5 kGray to 25 kGray, 5 kGray to 20 kGray, 5kGray to 15 kGray, and 5 kGray to 10 kGray.

(52) An injectable ganaxolone nanoparticulate formulation comprising:

(a) ganaxolone nanoparticles having a D50 of 2000 nm or less and (b) atleast one surface stabilizer;

wherein in comparative pharmacokinetic testing with an injectablenon-particulate ganaxolone formulation of the same dosage strength thenanoparticulate formulation exhibits a greater C_(max) than thenon-particulate ganaxolone formulation.

(53) An injectable ganaxolone nanoparticulate formulation comprising:

(a) ganaxolone nanoparticles having a D50 of 2000 nm or less and (b) atleast one surface stabilizer;

wherein in comparative pharmacokinetic testing with an injectablenon-particulate ganaxolone formulation of the same dosage strength thenanoparticulate formulation exhibits a greater brain AUC_(6 hrs) thanthe non-particulate ganaxolone formulation.

(54) An injectable ganaxolone nanoparticulate formulation comprising:

(a) ganaxolone nanoparticles having a D50 of 2000 nm or less and (b) atleast one surface stabilizer;

wherein in comparative pharmacokinetic testing with an injectablenon-particulate ganaxolone formulation of the same dosage strength thenanoparticulate formulation exhibits a greater brain concentration atany time from 15 to 100 minutes after administration than thenon-particulate ganaxolone formulation exhibits at the same time afteradministration.

(55). A method of treating a patient having a seizure disorder, stroke,or traumatic brain injury, the method comprising administering atherapeutically effective amount of the injectable neurosteroidformulation of any one of the preceding Specific Embodiments.

(56) The method of Specific Embodiment 55, wherein the seizure disorderis status epilepticus, refractory status epilepticus, super refractorystatus epilepticus, or PCDH19 female pediatric epilepsy.

(57) The method of Specific Embodiment 55 or 56 wherein the neurosteroidis ganaxolone or allopregnanolone and the dosage of neurosteroidadministered is from about 1 mg/kg to about 200 mg/kg.

(58) The method of Specific Embodiment of any one of Specific Embodiment55 to 57, wherein the neurosteroid is ganaxolone.

(59) The method of any one of Specific Embodiments 55 to 58 wherein theformulation is administered intravenously. The method of any one ofSpecific Embodiments 55 to 58 wherein the formulation is administeredintramuscularly.

(60) The method of Specific Embodiment 59 comprising administering asingle bolus dose of the formulation to the patient.

(61) The method of Specific Embodiment 60 wherein the single bolus doseprovides a sufficient amount of ganaxolone to provide a plasma C_(max)of ganaxolone of at least 1000 ng/mL in the patient.

(62) The method of Specific Embodiment 61, wherein the bolus doseprovides a sufficient amount of ganaxolone to provide a plasma C_(max)of ganaxolone of about 1000 ng/mL to about 6000 ng/mL in the patient.

(63) The method of Specific Embodiment 60 or 61, wherein the bolus doseis administered in less than 10 minutes and Cmax occurs within 1 hour ofcompletion of administration.

(64) The method of any one of Specific Embodiment 60 to 63, wherein thesingle bolus dose comprises from about 1 mg/kg to about 20 mg/kgganaxolone.

(65). The method of any one of Specific Embodiments 55 to 59 comprisingadministering multiple bolus doses of the ganaxolone formulation to thepatient.

(66) The method of Specific Embodiment 65 wherein the multiple bolusdoses are given over 1 to 10 days at intervals of 1 to 24 hours.

(67) The method of Specific Embodiment 65 wherein each bolus doseprovides a sufficient amount of ganaxolone to produce a plasma C_(max)of ganaxolone of at least 1000 ng/mL in the patient.

(68) The method of Specific Embodiment 65, wherein the interval betweenbolus doses is from about 10 to about 24 hours and once an initialC_(max) is reached the plasma concentration of ganaxolone is not below100 ng/mL at any time between bolus doses.

(69) The method of Specific Embodiment 65, wherein the interval betweenbolus doses is from about 20 to about 24 hours and once an initialC_(max) is reached the concentration of ganaxolone in the patient'splasma does not fall below 25% of the initial C_(max) at any timebetween bolus doses.

(70) The method of any one of Specific Embodiments 65 to 69 wherein eachbolus dose comprises about 1 mg/kg to about 20 mg/kg ganaxolone.

(71) The method of any one of Specific Embodiments 55 to 59 comprisingadministering an intravenous infusion of the ganaxolone formulation tothe patient, with or without an initial bolus dose.

(72) The method of Specific Embodiment 71 comprising administering theintravenous infusion for 1 to 10 consecutive days at a rate of 1 to 10mg/kg/hr without an initial bolus dose.

(73) The method of Specific Embodiment 72 comprising administering aninitial bolus dose of from about 1 mg/kg to about 20 mg/kg ganaxolone,followed within 24 hours by administration of an intravenous infusion ofthe ganaxolone formulation for 1 to 10 consecutive days at a rate of 1to 10 mg/kg/hr.

(74) The method of Specific Embodiment 73, wherein the initial bolusdose provides a sufficient amount of ganaxolone to provide an initialplasma C_(max) of ganaxolone of at least 1000 ng/mL in the patient andthe concentration of ganaxolone in the patient's plasma does not fallbelow 25% of the initial C_(max) until after the infusion is concluded.

(75) The method of Specific Embodiment 73, wherein the initial bolusdose provides a sufficient amount of ganaxolone to provide an initialplasma C_(max) of ganaxolone of about 100 ng/mL to about 8000 ng/mL inthe patient, the patient is then administered an intravenous infusion ofthe ganaxolone formulation at a constant dose sufficient to provide aconcentration of ganaxolone in the patient's plasma of at least 40% ofC_(max), followed by an intravenous infusion of ganaxolone formulationat a gradually reducing dose so that the concentration of ganaxolone inthe patient's plasma is less than 20% of C_(max) when the intravenousinfusion is concluded.

(76) The method any one of Specific Embodiments 55 to 75 wherein theganaxolone formulation is a first active agent and is administeredconcurrently or sequentially with at least one additional active agent.

(77) The method of Specific Embodiment 76 wherein the at least oneadditional active agent is an anticonvulsant or anesthetic/sedative.

(78) The method of Specific Embodiment 77 wherein the at least oneadditional active agent is an anticonvulsant chosen from a GABA_(A)receptor modulator, a sodium channel blocker, a GAT-1 GABA transportermodulator, a GABA transaminase modulator, a voltage-gated calciumchannel blocker, and a peroxisome proliferator-activated alphamodulator.

(79) The method of Specific Embodiment 77 wherein the at least oneadditional active agent is an anesthetic/sedative chosen from aninhalational anesthetics (including desflurane, enflurane, ethylchloride, halothane, isoflurane, methoxyflurane, sevoflurane, andtrichloroethylene), an intravenous, non-barbiturate anesthetics(including atracurium, cisatracurium, etodimidate, ketamine, propofol,and rocuronium), a barbiturate anesthetic (including amobarbital,methohexital, pentobarbital, phenobarbital, secobarbital, thiamylal, andthiopental), and a benzodiazepine anesthetic (including diazepam,flunitrazepam, lorazepam, and midazolam).

(80) The method of Specific Embodiment 79, wherein the additional activeagent is an anesthetic/sedative and the patient is given a sufficientdosage of the anesthetic/sedative to induce coma.

(81) The method of Specific Embodiment 80, wherein the additional activeagent is a barbiturate.

(82) The method of Specific Embodiment 81, wherein the additional activeagent is pentobarbital or thiopental.

(83) The method of Specific Embodiment 79, wherein the additional activeagent is propofol.

(84) The method of Specific Embodiment 76, wherein a first additionalactive agent is an anticonvulsant and a second additional active agentis an anesthetic/sedative.

(85) The method of Specific Embodiment 84, wherein the anticonvulsant iscarbamazepine, tiagabine, levetiracetam, lamotrigine, pregabalin,gabapentin, or phenytoin and the anesthetic/sedative is pentobarbital,thiopental, or propofol.

EXAMPLES Abbreviations ALLO Allopregnanolone GNX Ganaxolone

HES Hydroxy ethyl starch

Example 1. Preparation of Ganaxolone Nanosuspension (10% wt) Via WetBead Milling

An aqueous slurry (250 g) containing ganaxolone (25 g), hydroxyethylstarch (7.5 g), sodium deoxycholate (0.5 g) and 30% simethicone (1 drop)was milled using a Netzsch Mill (Minicer) with 0.3 mm YTZ beads (Yttriumstabilized grinding media, Tosoh Corporation, Japan, ZrO₂+HfO₂ (95 wt %(weight %)), Y₂O₂ (5 wt %)). Two additional portions of solid sodiumdeoxycholate (0.5 g each) were added at 100 and 130 min after millinghad started. The particle size of the milled slurry was measured using aHoriba LA-910 laser diffraction particle size analyzer. After 170minutes of milling, D50 was 192 nm (188 nm after 1 min sonication). Atthis point, milling was stopped and the milled slurry was kept at roomtemperature overnight. The next morning, milling was resumed until thetotal milling time had reached 320 minutes, at which point D50 was 167nm (169 nm after 1 min sonication). The D50 particle size was measuredon a Horiba 910 Laser Light Scattering instrument.

Example 2. Preparation of Ganaxolone Nanosuspension (20% wt) Via WetBead Milling

An aqueous slurry (250 g) containing ganaxolone (50 g), hydroxyethylstarch (15 g), sodium deoxycholate (3 g) and 30% simethicone (0.15 g)was milled using a Netzsch mill (Minicer) with 0.3 mm YTZ beads for 240minutes. The D50 of the milled slurry was 189 nm (185 nm after 1 minsonication).

Example 3. Preparation of Ganaxolone Nanosuspension (20% wt) Via WetBead Milling Using 0.2 Mm YTZ Beads

An aqueous ganaxolone slurry having the same composition as described inExample 2 was milled using a Netzsch mill (Minicer) with 0.2 mm YTZbeads for 245 minutes. The D50 was 172 nm (167 nm after 1 minutesonication).

Example 4. Preparation of Ganaxolone Nanosuspension Containing Dextran70 Via Wet Bead Milling

An aqueous ganaxolone slurry (250 g) containing ganaxolone (25 g),dextran 70 (7.5 g), sodium deoxycholate (1.5 g), and 30% simethicone(0.075 g) was milled using a Netzsch mill (Minicer) with 0.2 mm YTZbeads for 195 minutes to obtain a ganaxolone nanosuspension with D50 of159 nm (158 nm after 1 minute sonication). Prolonged milling causedparticle size to increase to 215 nm (212 nm after 1 min sonication).

Example 5. Preparation of Ganaxolone Nanosuspension Containing 10%Hydroxyethyl Starch

An aqueous ganaxolone suspension (250 g) containing ganaxolone (25 g),hydroxyethyl starch (25 g), sodium deoxycholate (3 g) and 30%simethicone (0.15 g) was milled using a Netzsch mill (Minicer) with 0.2mm YTZ beads for 150 minutes to obtain a ganaxolone nanosuspension withD50 value of 139 nm (140 nm after 1 minute sonication).

Example 6. Dilution of Ganaxolone Nanosuspension Concentrate and SterileFiltration Through 0.2 Micron Filter

The ganaxolone nanosuspension of Example 5 was diluted 5-fold with HPLCgrade water to obtain a nanosuspension containing about 20 mg/mLganaxolone. This suspension was filtered through a 0.2 um syringe filter(Cellulose acetate, 25 mm, 0.2 μm, product #: 13-250020-25 PK,Scientific Strategies). The particle size of the filtered ganaxolonesuspension was measured and found to be: D50, 143 nm (143 nm after 1minute sonication); D90, 219 nm; D95, 289 nm.

Example 7. Procedure for Freeze Drying Ganaxolone Nanosuspension

Ganaxolone nanosuspension, prepared according to the procedure ofExamples 1-5 (2 mL), was placed in a 20 mL HDPE scintillation vialfollowed by addition of appropriate amount of solid inactivepharmaceutical excipients. After the solid excipients were dissolved byvisual inspection, the vial was immersed in a dry ice acetone bath untilthe content in the vial was completely frozen. Solid excipients include,for example, sucrose, mannitol, dextrose, lactose, D-sorbitol, and NaCl.

The vial was then placed in a freeze dryer flask for lyophilization andlyophilized until a dry solid was obtained. The lyophilized powder wasre-dispersed in either water or 0.9% saline prior to particle sizemeasurement. Table 1 shows lyophilized ganaxolone formulationscontaining hydroxyethyl starch (ganaxolone/hydroxyethyl starch=3.3:1).The D50 values of the Table 1 formulations prior to freeze drying werebetween 214-230 nm. Table 2 shows lyophilized ganaxolone formulationscontaining dextran 70 (ganaxolone/dextran 70=3.3:1). The D50 value priorto freeze-drying of the ganaxolone nanosuspension with sucrose was 0.212μm (microns) prior to freeze drying. Table 3 shows lyophilizedganaxolone formulations containing hydroxyethyl starch(ganaxolone/hydroxyethyl starch=1:1). The D50 value prior to freezedrying was 0.139 μm.

TABLE 1 Particle size values (D50) of freeze dried ganaxolonenanosuspension formulations (ganaxolone to hydroxyethyl starch 130/0.4ratio is 3.3:1) after redispersion in water Composition (wt %)Formulation A B C D E F G Ganaxolone 73.48 53.74 42.36 53.74 42.36 53.7442.36 Hydroxyethyl 22.04 16.12 12.71 16.12 12.71 16.12 12.71 starchSodium 4.41 3.22 2.54 3.22 2.54 3.22 2.54 deoxycholate Simethicone 0.070.05 0.04 0.05 0.04 0.05 0.04 (30%) Sucrose 0.00 26.87 42.36 0.00 0.000.00 0.00 Mannitol 0.00 0.00 0.00 26.87 42.36 0.00 0.00 dextrose 0.000.00 0.00 0.00 0.00 26.87 42.36 lactose 0.00 0.00 0.00 0.00 0.00 0.000.00 D-Sorbitol 0.00 0.00 0.00 0.00 0.00 0.00 0.00 NaCl 0.00 0.00 0.000.00 0.00 0.00 0.00 Total 100.00 100.00 100.00 100.00 100.00 100.00100.00 D50 (μm)^(a) 24.168 0.320 0.279 51.85 20.48 0.330 11.223 (3.518)(0.225) (0.209) (6.510) (3.36) (0.252) (4.688) Composition (wt %)Formulation H I J K L M Ganaxolone 53.74 42.36 53.74 42.36 40.64 29.75Hydroxyethyl 16.12 12.71 16.12 12.71 12.19 8.93 starch Sodium 3.22 2.543.22 2.54 2.44 1.79 deoxycholate Simethicone 0.05 0.04 0.05 0.04 0.040.03 (30%) Sucrose 0.00 0.00 0.00 0.00 40.64 59.51 Mannitol 0.00 0.000.00 0.00 0.00 0.00 dextrose 0.00 0.00 0.00 0.00 0.00 0.00 lactose 26.8742.36 0.00 0.00 0.00 0.00 D-Sorbitol 0.00 0.00 26.87 42.36 0.00 0.00NaCl 0.00 0.00 0.00 0.00 4.06 0.00 Total 100.00 100.00 100.00 100.00100.00 100.00 D50 (μm)^(a) 0.352 0.971 0.328 0.335 0.226 0.207 (0.245)(0.418) (0.216) (0.227) (0.199) (0.208) ^(a)D50 values in parenthesisare after 1 minute sonication.

TABLE 2 Particle size values (D50) of freeze dried ganaxolonenanosuspension formulations containing dextran 70 after redispersion inwater Composition (wt %) Formulation A (no sucrose) B (with sucrose)Ganaxolone 73.48 18.65 Dextran 70 22.04 5.60 Sodium deoxycholate 4.411.12 30% Simethicone 0.07 0.02 emulsion Sucrose 0.00 74.61 Total 100.00100.00 D50 (μm)^(a) 26.165 (3.793) 0.224 (0.224) ^(a)D50 values inparenthesis are after 1 minute sonication.

TABLE 3 Particle size values (D50) of freeze dried ganaxolonenanosuspension formulations (ganaxolone to hydroxyethyl starch 130/0.4ratio is 1:1) after redispersion in 0.9% saline for injectionComposition (wt %) Formulation A (no sucrose) B (with sucrose)Ganaxolone 47.13 24.26 hydroxyethyl starch 47.13 24.26 Sodiumdeoxycholate 5.66 2.91 30% Simethicone 0.08 0.04 emulsion Sucrose 0.0048.52 Total 100.00 100.00 D50 (μm)^(a) 0.275 (0.174) 0.161 (0.150)^(a)D50 values in parenthesis are after 1 minute sonication.

Example 8. Particle Size Storage Stability of Ganaxolone NanosuspensionContaining 20% Ganaxolone, 6% Hydroxyethyl Starch, 1.2% SodiumDeoxycholate and 0.06% Simethicone (30% Emulsion)

A ganaxolone nanosuspension containing (wt %) 20% ganaxolone, 6%hydroxyethyl starch, 1.2% sodium deoxycholate, and 0.6% simethicone (30%emulsion) was prepared by the procedure described in Example 2. The D50particle size was measured on a Horiba 910 Laser Light Scatteringinstrument over a 17 day period. Initial particle size was approximately189 nm. Particle size initially increased about 10% but remained stableafter the initial increase from the remainder of the 17 day period. SeeFIG. 1 .

Example 9. Ganaxolone Nano Suspension Containing Poloxamer 188

A KDL Bachofen Mill was configured with the batch chamber attachment(approx. 350 ml) and the 96 mm polyurethane rotor attached to the shaft.Next, 265 ml of 0.3 mm ytria-zirconia beads were added dry to thechamber, followed by 176.7 gm of the Ganaxolone (GNX) slurry. Slowly,over 15 minutes, the ganaxolone slurry was added to the milling mediacontaining Pluronic F-68 (Poloxamer 188) with sustained stirring. Themixture was stirred slowly overnight. The slurry was milled at Speed 1(1500 rpm) with intermittent measurement of particle size. After 90 min,the D50 particle size was determined to be 378 nm. The D50 measurementwas measured on a Horiba 910 Laser Light Scattering instrument.

Milling Media Pluronic F-68 27.0 g Sodium deoxycholate 2.7 g Simethiconeemulsion 30% 0.2 g Water (DI) to 200 g Ganaxolone Slurry Ganaxolone 50 gMilling Media 150 g Final Milling Composition (wt %) Ganaxolone 25%Pluronic F-68 10% Deoxycholate  1%

Example 10. Ganaxolone Nano Suspension Containing Poloxamer 188, 0.1 mmBeads

The KDL Bachofen mill was configured with the batch chamber attachment(approx. 350 ml) and the 96 mm polyurethane rotor attached to the shaft.Next, 300 ml of 0.1 mm ytria-zirconia beads were added dry to thechamber, followed by 134.6 gm Ganaxolone (GNX) slurry having thecomposition given in preceding Example 9. The slurry was milled for 60minutes and the D50 particle size was measured after 20, 40, 60 minutesof milling.

Time (min) Particle size, μm After sonication, μm 20 0.182 0.183 400.164 0.165 60 0.162

Example 11. Ganaxolone Nano Suspension Containing 12.5% Poloxamer 188and Dextran

The KDL Bachofen mill was configured with the batch chamber attachment(approx. 350 ml) and the 96 mm polyurethane rotor attached to the shaft.Next, 300 ml of 0.1 mm yttria-zirconia beads were added dry to thechamber, followed by 176.5 gm of the Ganaxolone (GNX) millingsuspension. The ganaxolone milling suspension was prepared by combiningthe dextran, Pluronic F-68, sodium deoxycholate, and simethiconeemulsion ingredients with stirring, and then adding the ganaxolone lastwith stirring. The suspension stirred for 1.5 hr. The suspension (176.5gm was added to the batch chamber and the mill started at Speedsetting 1. The slurry was milled for 60 minutes and the D50 particlesize was measured after 20, 40, 50, and 60 minutes of milling.

Ganaxolone Milling Suspension Dextran (40K mol. wt.) 10.0 g PluronicF-68 25.0 g Sodium deoxycholate  0.5 g Simethicone emulsion 30%  0.2 gGanaxolone 20.0 g Water (DI) to 200 g Final Milling Composition (wt %)Ganaxolone 20% Dextran  5% Pluronic F-68 25% Sodium Deoxycholate 0.25% 

Time (min) Particle size, μm After sonication, μm 20 0.221 0.219 400.173 — 50 0.166 0.168 60 0.164 —

The milled suspension above (64.4 gm) was treated with methyl paraben Na(0.074 gm and citric acid (0.027 gm) and the particle size monitoredover time. The results are presented graphically in FIG. 2 .

Day Particle size, μm 0 0.191 2 0.194 5 0.313 6 0.317

Example 12. Preparation of Allopregnanolone Nanosuspension Via Wet BeadMilling

An aqueous slurry (125 g) containing allopregnanolone (12.5 g),hydroxyethyl starch (12.5 g), sodium deoxycholate (1.5 g) and 30%simethicone emulsion (0.075 g) was milled using a Netzsch mill (Minicer)with 0.2 mm YTZ beads for 210 minutes. The D50 of the milled slurry was96 nm (96 nm after 1 min sonication). Particle size distribution plotsfor ganaxolone and allopregnanolone formulations prepared as describedin Examples 12-15 are provided in FIGS. 6A-6D.

Example 13. Preparation of Allopregnanolone Nanosuspension Via Wet BeadMilling

An aqueous slurry (169.7 g) containing allopregnanolone (21.5 g),hydroxyethyl starch 130/0.4 (26.5 g), sodium deoxycholate (2.1 g) and30% simethicone emulsion (0.10 g) was milled using a Netzsch mill(Minicer) with 0.2 mm YTZ beads for 240 minutes. The D50 of the milledslurry was 98 nm (97 nm after 1 min sonication).

Example 14. Preparation of Ganaxolone Nanosuspension Containing PovidoneVia Wet Bead Milling

An aqueous ganaxolone slurry (175 g) containing ganaxolone (17.5 g),povidone (17.5 g), sodium deoxycholate (2.1 g), and 30% simethicone(0.105 g) was milled using a Netzsch mill (Minicer) with 0.2 mm YTZbeads for 180 minutes to obtain a ganaxolone nanosuspension with D50 of109 nm (111 nm after 1 minute sonication). The D50 value was 114 nm (113nm after 1 minute sonication) after 3 days of storage at ambientconditions.

Example 15. Preparation of Ganaxolone Nanosuspension ContainingHydroxyethyl Starch Via Wet Bead Milling

An aqueous ganaxolone slurry (175 g) containing ganaxolone (17.5 g),hydroxyethyl starch 130/0.4 (17.5 g), sodium deoxycholate (2.1 g), and30% simethicone (0.105 g) was milled using a Netzsch mill (Minicer) with0.2 mm YTZ beads for 240 minutes to obtain a ganaxolone nanosuspensionwith D50 of 106 nm (107 nm after 1 minute sonication).

Example 16. Bioavailability of Nanosuspension and Captisol Formulations

The ganaxolone concentration in rat plasma and rat brain followingadministration of 9, 12, or 15 mg/kg ganaxolone as a Captisol solutionor hydroxyethyl starch 130/0.4 nanosuspension was determined. MaleSprague-Dawley rats, 8-9 weeks of age, from Harlan Labs were used.Animals received food and water ad libitum throughout the study and weremaintained on a 12 hr/12 hr light dark schedule with lights on at 7:00AM. Animals were weighed prior to compound administration. Ganaxolonesolutions were formulated at 2.5 mg/mL and the volume was adjusted toaccommodate larger dosages. Injections were administered via the tailvein as a bolus dose.

Plasma was collected and 5, 15, 30, 60 or 120 minutes post dosing.Brains were collected at 5, 30, and 120 minutes post dosing. Three ratswere used for each time point, and the reported ganaxolone levels arethe mean of ganaxolone plasma or brain levels of all three rats. Bloodwas collected by retro-orbital bleed or cardiac puncture. Blood sampleswere collected into K²⁺EDTA coated tubes. Plasma samples were preparedby spinning blood in a refrigerated centrifuge (3000 rpm for 10 min at4° C.). Plasma PK characteristics were similar for the ganaxoloneCaptisol and nanosuspension formulations (see FIGS. 3A-3C), however thenanosuspension produced significantly higher and longer lasting brainganaxolone levels (See FIGS. 4A-4C). The ganaxolone Captisol andnanosuspension formulations used in this experiment and the experimentpresented in the next example are provided in TABLE 4A.

TABLE 4A Ganaxolone Concentration Formulation Composition (wt %) (mg/mL)Ganaxolone/ Ganaxolone: 0.22% 2.5 mg/mL Captisol Captisol: 26.84%solution Water: 72.94% Ganaxolone Ganaxolone: 0.25% 2.5 mg/mLNanosuspension Hydroxyethyl Starch 130/0.4: 0.25% Sodium Deoxycholate:0.03% Simethicone, 30% emulsion: 0.0006% Water: 99.47%

Example 17. Brain and Plasma Levels of Ganaxolone and AllopregnanoloneNanosuspension and Captisol Solutions

Rats were dosed with approximately 1 mg/ml ganaxolone as ananosuspension or as a Captisol solution. A dosage of 1 mg/kg was used.The ganaxolone plasma levels and brain levels were determined at 5, 15,30, and 120 minutes. Three rats were used for each time point, and thereported ganaxolone levels are the mean of ganaxolone plasma or brainlevels of all three rats. The same study was conducted usingallopregnanolone, as a nanosuspension or Captisol solution. Theganaxolone and allopregnanolone formulations used in this experiment aregiven in TABLE 4B. The results of this experiment are shown in FIGS.5A-5B.

TABLE 4B Drug Concentration Formulation Composition (wt %) (mg/mL)Ganaxolone/ Ganaxolone: 0.097% 0.99 mg/ml  Captisol Captisol: 5.85%solution Water: 94.05% Ganaxolone Ganaxolone: 0.10% 0.98 mg/mLNanosuspension Hydroxyethyl Starch 130/0.4: (D50: 143 nm) 0.10% SodiumDeoxycholate: 0.012% Simethicone, 30% emulsion: 0.0006% Water: 99.79%Allopregnanolone/ Ganaxolone: 0.079% 0.81 mg/mL Captisol solutionCaptisol: 5.85% Water: 94.07% Allopregnanolone Allopregnanolone: 0.10%0.95 mg/mL nanosuspension Hydroxyethyl Starch 130/0.4: (D50: 95 nm)0.10% Sodium Deoxycholate: 0.012% Simethicone, 30% emulsion: 0.0006%Water: 99.79%

Example 18. Behavioral Observations and Sedation Levels of GanaxoloneNanosuspension and Captisol Solutions

This study consisted of administering ganaxolone at 9, 12, or 15 mg/mLor vehicle (negative control) in the Captisol and nanosuspensionformulations. Injections were administered via the tail vein as a bolusdose. The behavior of the animals was recorded at 5, 15, 30, 60, 120,180, and 240 minutes post dosing. Terminal blood/plasm and brain sampleswere collected at 4 hours.

Animals are as described in Example 16. Four animals were used for eachtreatment group. TABLE 5 below illustrates the experimental design forthe sedation experiments. The formulations are described in Example 16,TABLE 4A. For each of the experimental conditions listed in TABLE 5 theevaluation and endpoint is (1) Sedation level and duration and (2)ganaxolone level in plasma and brain at experiment termination (4 hoursafter dosing).

TABLE 5 Treatment Formulation Group Size Dose (mg/kg) Vehicle 30%Captisol 4 0 Ganaxolone 30% Captisol 4 9 Ganaxolone 30% Captisol 4 12Ganaxolone 30% Captisol 4 15 Vehicle Nanosuspension 4 0 GanaxoloneNanosuspension 4 9 Ganaxolone Nanosuspension 4 12 GanaxoloneNanosuspension 4 15

Rats were observed for behavioral changes at 5, 15, 30, 60, 120, 180,and 240 minutes post administration. The observer was blinded totreatment. Qualitative behavioral changes were scored as follows alongwith any relevant observations.

0=awake, absence of sedation; no change in observed locomotion orbehavior

1=light sedation; impaired; slowed movement, unresponsive to somestimuli, intact righting reflex.

2=deep sedation; sedated; lying on side, loss of righting reflex (LRR)

3=anesthesia; loss of toe-pinch reflex.

The health of the animals was monitored, particularly body temperature.If animals were cold to the touch, core body temperature was monitoredby rectal probe and recorded. However, a heating pad not needed tomaintain body temperature. Animals that received a sedation behaviorscore of 2 were placed on a blue pad lying on top of the bedding toprevent choking and these animals were closely monitored. All atypicalor abnormal behavior or health issues were documented.

The behavior was scored using a four point scale (0, 1, 2, or 3) and thecategorical data was analyzed by non-parametric Kruskal-Wallis ANOVA ateach individual time point using Prism GraphPad (version 6). Post-hocanalysis consisted of Dunn's multiple (all pairwise) comparison tests,with significance set at P<0.05. PK data was analyzed by two-way ANOVA.

Both the Captisol and nanosuspension ganaxolone formulations were highlysedating and all rats at every dose-level received a sedation score of 2at 5 min post injection (FIGS. 7A-7C). The formulations exhibiteddose-related effects on sedation as time progressed, with sedationlasting from 30-120 min, depending on dose and vehicle formulation(FIGS. 7A-7C). The most sedating dose/formulation combination was the 15mg/kg nanosuspension ganaxolone formulation; 2 of the 4 rats in thisgroup were still sedated at 120 min following injection. All of theanimals regardless of dose or formulation were awake 3 hours followinginjection.

In general, the nanosuspension formulation produced a longer duration ofsedation than the Captisol formulation. Individual Kruskal-Wallisnon-parametric ANOVA's at each time point did not reveal any statisticaldifferences between the Captisol and nanosuspension formulations.However, when analyzed by Mann-Whitney non-parametric t-test, theganaxolone dose of 12 mg/kg in Captisol was significantly less sedatingthan the comparable nanosuspension ganaxolone dose at 60 min postadministration.

The nanosuspension formulations were found to have increased behavioraleffects as noted by: longer latency to wake-up, hemolysis/bloody urine(2 rats at 12 mg/kg and 1 rat at 15 mg/kg) and slowed/irregularbreathing immediately following injection (1 rat at 12 mg/kg and 1 ratat 15 mg/kg).

Plasma PK characteristics were similar between the two formulations.However, when examining brain levels the nanosuspension formulationproduced significantly higher and longer lasting levels than theCaptisol formulation. This brain PK paralleled the behavioral sedativeresponse. These data are shown in FIGS. 3 and 4 .

In summary, both the Captisol and nanosuspension formulations werehighly sedating and exhibited dose-related effects on sedation as afunction of time. The nanosuspensions appeared to have increasedsedation. This increased behavioral response was most likely produced byhigher neurosteroid brain absorption of the nanosuspension formulation.

Example 19. Ebeam Irradiation of Injectable Nanoparticle Formulations

Injectable neurosteroid nanoparticle formulations, prepared as describedin the preceding examples were filled into 8 ml glass vials and capped.The vials were subjected to a 25 kGy dose of ebeam irradiation, astandard dose for producing sterile product. The nanoparticleformulations were assessed for appearance before and after ebeamirradiation, HPLC assay and impurity profiles before and afterirradiation, relative viscosity before and after irradiation, andparticle size (D50 and D90) before and after ebeam irradiation, Nochange in appearance was observed after ebeam irradiation for any of theneurosteroid nanosuspension tested. Neurosteroid assay and impurityprofiles were determined via standard HPLC procedures. Viscositymeasurements were obtained using an Ostwald viscometer. Relativeviscosity was calculated as an efflux time ratio between nanosuspensionand deionized water. D50 and D90 particle size measurements obtainedusing a Horiba 910 Laser Light Scattering instrument.

TABLE 6 shows the compositions of formulation I-VI which are used in theebeam experiments that follow. The polymers used in formulation I-VI areI, Plasdone C-17; II, V, and VI, hydroxyethyl starch 130/0.4; III,Dextran 70; IV, Plasdone C-12; The API is ganaxolone for allformulations except formulation VI. The API is allopregnanolone forformulation VI.

TABLE 6 Composition of formulations I-VI Formulation I II III IV V VI*Ganaxolone 5.43% 5.42% 5.57% 5.50% 5.50% 5.50% Polymer 5.43% 5.42% 5.57%5.50% 11.00% 5.50% Sodium Deoxycholate 0.65% 0.65% 0.67% 0.66% 0.66%0.66% Simethicone 30% emulsion 0.03% 0.03% 0.03% 0.03% 0.03% 0.03%Deionized water 88.46% 88.46%  88.16% 88.31% 82.81% 88.31% Total 100.00% 100% 100.00% 100.00% 100.00% 100.00% *API was allopregnanolone

TABLE 7 shows the HPLC assay of the neurosteroid and impurity profilefor various injectable neurosteroid nanoparticle formulations (I-VI)after ebeam irradiation at 25 KGy (kilo gray). No significant change inimpurity profile was observed for any tested formulation.

TABLE 7 HPLC assay (% Impurity profile Polymer to control) after afterebeam @25 Formulation neurosteroid ratio ebeam@25 KGy KGy I 1 100.40 Nochange II 1 101.49 No change III 1 99.22 No change IV 1 98.01 No changeV 2 99.56 No change VI 1 99.93 No change

TABLE 8 shows the D50 and D90 values for the above injectableneurosteroid nanoparticle formulations (I-VI) before and after ebeamirradiation. Samples contained sucrose at two times the weight percentof neurosteroid.

TABLE 8 Unirradiated After ebeam irradition@25 KGy control No sucrosewith Sucrose (2 × neurosteroid) Formulation D50 (nm) D90 (nm) D50 (nm)D90 (nm) D50 (nm) D90 (nm) I 168 355 178 415 178 402 II 163 273 171 299180 383 III 150 231 156 236 163 267 IV 153 218 155 215 151 215 V 155 238162 271 170 323 VI 107 164 107 166 106 164

TABLE 9 presents relative viscosity for ganaxolone formulations I-Vlisted in TABLE 6 before and after ebeam irradiation.

TABLE 9 Relative viscosity Relative viscosity before ebeam (after ebeamFormulation control @25 KGy) % change I 2.226 2.279 2.42% II 3.178 2.850−10.32% III 4.508 3.428 −23.96% IV 1.853 1.856 0.16% V 6.300 5.827−7.51%

Example 20. Ebeam Irradiation of Lyophilized Powder of GanaxoloneNanoparticle Formulations

Sucrose (250 mg) was added to the liquid ganaxolone nanoparticleformulation (2 ml) in an 8 ml glass vial and dissolved. The mixture wasfrozen on dry ice for about 2 hours and lyophilized to obtain a whitecake. The compositions of the lyophilized powders are shown in Table 10.The lyophilized powders were subjected to 25 KGy ebeam irradiation. Theparticle size data were obtained after reconstituting the lyophilizedformulations in deionized water.

TABLE 10 Composition, physical and chemical stability of lyophilizedganaxolone nanoparticle powder after ebeam irradiation at a dose of 25KGy Lyophilized Lyophilized powder II Lyophilized powder I (Hydroxyethylpowder III (Plasdone C-17) starch 130/0.4) (dextran 70) Ingredients % wt% wt % wt Ganaxolone 22.58% 22.56% 22.88% Polymer 22.58% 22.56% 22.88%Na Deoxycholate 2.70% 2.71% 2.75% Simethicone 0.14% 0.12% 0.12% Sucrose51.99% 52.04% 51.36% Total 100.00% 100.00% 100.00% D50 (nm) 150 188 147D90 (nm) 266 455 259 HPLC assay 97.6% 97.6% 95.4% Impurity profile Nochange No change No Change

What is claimed is:
 1. An injectable neurosteroid formulation comprising nanoparticles having a D50 of less than 2000 nm, the nanoparticles comprising a) a neurosteroid of the Formula I:

or a pharmaceutically acceptable salt thereof, wherein: X is O, S, or NR¹⁰; R¹ is hydrogen, hydroxyl, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted aryl, or optionally substituted arylalkyl; R⁴ is hydrogen, hydroxyl, oxo, optionally substituted alkyl, or optionally substituted heteroalkyl, R², R³, R⁵, R⁶, and R⁷ are each independently hydrogen, hydroxyl, halogen, optionally substituted alkyl, or optionally substituted heteroalkyl; R⁸ is hydrogen or alkyl and R⁹ is hydroxyl; or R⁸ and R⁹ are taken together to form an oxo group; R¹⁰ is hydrogen, hydroxyl, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted aryl, or optionally substituted arylalkyl where each alkyl is a C₁-C₁₀alkyl, C₃-C₆cycloalkyl, (C₃-C₆cycloalkyl)C₁-C₄alkyl, and optionally contains a single bond replaced by a double or triple bond; each heteroalkyl group is an alkyl group in which one or more methyl group is replaced by an independently chosen —O—, —S—, —N(R¹⁰)—, —S(═O)— or —S(═O)₂—, where R¹⁰ is hydrogen, alkyl, or alkyl in which one or more methylene group is replaced by —O—, —S—, —NH, or —N— alkyl; and b) at least one surface stabilizer.
 2. The injectable neurosteroid formulation of claim 1, comprising nanoparticles having a D50 of less than 500 nm; wherein the formulation is an intravenous formulation; and the neurosteroid is ganaxolone.
 3. The injectable neurosteroid formulation of claim 1 or 2, wherein the at least one surface stabilizer is a polymeric surface stabilizer and the polymeric surface stabilizer is hydroxyethyl starch, dextran, povidone, or a mixture of any the foregoing.
 4. The injectable neurosteroid formulation of any one of claims 1 to 3, wherein the formulation comprises an additional surface stabilizer and the additional surface stabilizer is an ionic or nonionic surfactant; and an antifoaming agent.
 5. The injectable neurosteroid formulation of claim 4, wherein the polymeric surface stabilizer is hydroxyethyl starch; the surfactant is sodium cholate, sodium deoxycholate, sodium cholesterol sulfate, or a mixture of any of the foregoing; and the antifoaming agent is simethicone.
 6. The injectable neurosteroid formulation of any one of claims 1 to 5 additionally comprising a cryoprotectant, wherein the cryoprotectant is sucrose, dextrose, lactose, D-sorbitol, or a mixture of any of the foregoing.
 7. The injectable neurosteroid formulation of any one of claims 1 to 6 additionally comprising one or more of the following (a) 0.5% to 1.5% sodium chloride (weight percent); (b) a buffer; (c) a preservative, wherein the preservative is benzyl alcohol, chlorbutanol, 2-ethoxyethanol, parabens (including methyl, ethyl, propyl, butyl, and combinations), benzoic acid, sorbic acid, chlorhexidene, phenol, 3-cresol, thimerosal, a phenylmercurate salt, or a mixture of any of the foregoing.
 8. The injectable neurosteroid formulation of claim 1, wherein the neurosteroid is ganaxolone or allopregnanolone; the at least one surface stabilizer is a polymeric surface stabilizer selected from hydroxyethyl starch, dextran, povidone, and a mixture of any of the foregoing, wherein the (wt:wt) ratio of the neurosteroid to the polymeric surface stabilizer is about 4:1 to about 0.5:1; and the formulation comprises an additional surface stabilizer and the additional surface stabilizer is a surfactant, selected from sodium deoxycholate, sodium cholesterol sulfate, and a mixture of any of the foregoing; wherein the ratio of neurosteroid to surfactant (w:w) is about 10:1.5 to about 10:0.1.
 9. The formulation of any one of claims 1 to 8, wherein the formulation is in the form of a lyophilized powder.
 10. The formulation any one of claims 1 to 9, wherein the formulation is an aqueous suspension and the neurosteroid concentration is about 0.1 mg/mL to about 300 mg/mL.
 11. The injectable neurosteroid formulation of claim 1, wherein the formulation is an aqueous formulation comprising (a) nanoparticles having a D50 of less than 500 nm, the nanoparticles comprising ganaxolone, wherein the weight percent of the ganaxolone is 1 to 10%; (b) a polymeric surface stabilizer selected from hydroxy ethyl starch, dextran, and povidone, wherein the weight percent of the polymeric surface stabilizer is 2 to 20%; (c) an additional surface stabilizer wherein the additional surface stabilizer is an ionic or nonionic surfactant selected from sodium cholate, sodium deoxycholate, and sodium cholesterol sulfate, wherein the weight percent of surfactant is 0.1% to 2.0%; and (d) an antifoaming agent.
 12. The injectable neurosteroid formulation of claim 1 wherein the formulation is an aqueous formulation comprising (a) nanoparticles having a D50 of less than 500 nm, the nanoparticles comprising ganaxolone, wherein the weight percent of the ganaxolone is about 5%; (b) a polymeric surface stabilizer selected from hydroxy ethyl starch 130/0.4 and plasdone C-12, wherein the weight percent of the polymeric surface stabilizer is about 5% to about 10%; (c) an additional surface stabilizer wherein the additional surface stabilizer is sodium deoxycholate, wherein the weight percent of sodium deoxycholate is about 0.75%; and (d) simethicone, wherein the weight percent of simethicone is 0.009%.
 13. A method for sterilizing the injectable neurosteroid nanoparticle formulation of any one of claims 1 to 12, comprising subjecting the formulation to ebeam radiation, wherein the method produces a sterilized neurosteroid nanoparticle formulation containing a degradant concentration of not more than 0.2% w/w of neurosteroid.
 14. The injectable neurosteroid formulation of any one of claims 1 to 12, wherein the formulation has been sterilized by ebeam irradiation and wherein the formulation contains a degradant concentration of not more than 0.2% w/w of the neurosteroid.
 15. The injectable neurosteroid formulation of claim 14, wherein the ebeam irradiation is a cumulative dose of about 25 kGray.
 16. An injectable ganaxolone nanoparticulate formulation comprising: (a) ganaxolone nanoparticles having a D50 of 2000 nm or less and (b) at least one surface stabilizer; wherein in comparative pharmacokinetic testing with an injectable non-particulate ganaxolone formulation of the same dosage strength the nanoparticulate formulation exhibits a greater Cmax than the non-particulate ganaxolone formulation.
 17. An injectable ganaxolone nanoparticulate formulation comprising: (a) ganaxolone nanoparticles having a D50 of 2000 nm or less and (b) at least one surface stabilizer; wherein in comparative pharmacokinetic testing with an injectable non-particulate ganaxolone formulation of the same dosage strength the nanparticulate formulation exhibits a greater brain concentration at any time from 15 to 100 minutes after administration than the non-particulate ganaxolone formulation exhibits at the same time after administration.
 18. A method of treating a patient having a seizure disorder, stroke, or traumatic brain injury, the method comprising administering intravenously a therapeutically effective amount of the injectable neurosteroid formulation of any one of claims 1 to 17, wherein the neurosteroid is ganaxolone.
 19. The method of claim 18, wherein the seizure disorder is status epilepticus, refractory status epilepticus, super refractory status epilepticus, or PCDH19 female pediatric epilepsy.
 20. The method of claim 18 wherein the dosage of ganaxolone administered is from about 1 mg/kg to about 200 mg/kg.
 21. The method of claim 18 or 19 comprising administering a single bolus dose of the formulation to the patient; wherein the single bolus dose provides a sufficient amount of ganaxolone to provide a plasma C_(max) of ganaxolone of at least 1000 ng/mL in the patient.
 22. The method of claim 18 or 19 comprising administering multiple bolus doses of the ganaxolone formulation to the patient, wherein the multiple bolus doses are given over 1 to 10 days at intervals of 1 to 24 hours, wherein each bolus dose provides a sufficient amount of ganaxolone to produce a plasma C_(max) of ganaxolone of at least 1000 ng/mL in the patient.
 23. The method of claim 18 or 19 comprising administering an intravenous infusion of the ganaxolone formulation to the patient, with or without an initial bolus dose, for 1 to 10 consecutive days at a rate of 1 to 10 mg/kg/hr without an initial bolus dose.
 24. The method of claim 23 comprising administering an initial bolus dose of from about 1 mg/kg to about 20 mg/kg ganaxolone, followed within 24 hours by administration of an intravenous infusion of the ganaxolone formulation for 1 to 10 consecutive days at a rate of 1 to 10 mg/kg/hr; sufficient amount of ganaxolone to provide an initial plasma C_(max) of ganaxolone of at least 1000 ng/mL in the patient and the concentration of ganaxolone in the patient's plasma does not fall below 25% of the initial C_(max) until after the infusion is concluded.
 25. The method any one of claims 18 to 24 wherein the injectable ganaxolone formulation is a first active agent and is administered concurrently or sequentially with at least one additional active agent; and the at least one additional active agent is an anticonvulsant or anesthetic/sedative. 